U.S. patent application number 15/655664 was filed with the patent office on 2018-02-08 for novel scaffolds for intracellular compound delivery for the detection of cancer cells.
The applicant listed for this patent is Indicator Systems International, Inc., ISI Life Sciences Inc.. Invention is credited to John FANTE, Theodore HESSLER, III, Craig KESHISHIAN, Robert MORIARTY, Richard PARIZA, Gerald F. SWISS, David WHITE.
Application Number | 20180036312 15/655664 |
Document ID | / |
Family ID | 61071668 |
Filed Date | 2018-02-08 |
United States Patent
Application |
20180036312 |
Kind Code |
A1 |
SWISS; Gerald F. ; et
al. |
February 8, 2018 |
Novel Scaffolds for Intracellular Compound Delivery for the
Detection of Cancer Cells
Abstract
Disclosed are folic acid or pteroic acid conjugates and methods
for using such conjugates. The conjugates described herein comprise
a chemotherapeutic drug or imaging agent covalently bound thereto
through a bond or via a linker that is intracellularly disrupted
by, e.g., endogenous enzymes such as esterases, lipases and the
like so as to provide for the chemotherapeutic drug free of the
folic or pteroic acid.
Inventors: |
SWISS; Gerald F.; (Newport
Beach, CA) ; MORIARTY; Robert; (Newport Beach,
CA) ; PARIZA; Richard; (Newport Beach, CA) ;
WHITE; David; (Newport Beach, CA) ; FANTE; John;
(Newport Beach, CA) ; HESSLER, III; Theodore;
(Newport Beach, CA) ; KESHISHIAN; Craig; (Newport
Beach, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
ISI Life Sciences Inc.
Indicator Systems International, Inc. |
Newport Beach
Newport Beach |
CA
CA |
US
US |
|
|
Family ID: |
61071668 |
Appl. No.: |
15/655664 |
Filed: |
July 20, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15595930 |
May 15, 2017 |
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15655664 |
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15595920 |
May 15, 2017 |
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15595930 |
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PCT/US2017/013531 |
Jan 13, 2017 |
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15595920 |
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62393524 |
Sep 12, 2016 |
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62378128 |
Aug 22, 2016 |
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62370130 |
Aug 2, 2016 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 49/006 20130101;
A61K 31/4985 20130101; C07D 493/10 20130101; A61K 31/166 20130101;
A61K 49/0052 20130101; A61K 31/519 20130101; A61K 49/0043 20130101;
A61K 49/0076 20130101; A61K 49/0041 20130101; A61K 49/0071
20130101; A61K 31/136 20130101; G01N 33/574 20130101 |
International
Class: |
A61K 31/519 20060101
A61K031/519; A61K 31/166 20060101 A61K031/166; A61K 31/136 20060101
A61K031/136; A61K 31/4985 20060101 A61K031/4985 |
Claims
1. A conjugate that comprises: a targeting agent that targets the
conjugate to cancer cells and where the conjugate is subsequently
absorbed by said cancer cells wherein said conjugate targets said
cancer cells at a rate of at least 2:1 greater than it targets
non-cancerous cells; and a masked fluorescent imaging agent wherein
the masking group is covalently attached to the imaging agent
provided that the imaging agent is not a fluorescent-fluorescent
quencher pair and wherein the targeting agent is covalently bound
to a masked fluorescent imaging agent via a bond or a linker; and
further wherein, upon absorption of the conjugate by the cancer
cell, the masked fluorescent imaging agent is unmasked to permit
the imaging agent to be capable of producing a detectible
fluorescent signal.
2. The conjugate of claim 1 wherein the linker is a oxyalkylene
linker comprising from 1 to oxyalkylene units.
3. A conjugate of the formula IA and IB: ##STR00088## where L is a
linker, X is a masked imaging agent, and Y is --O-- or >NR.sup.1
where R.sup.1 is hydrogen, C.sub.1-C.sub.4 alkyl, substituted
C.sub.1-C.sub.4 alkyl; C.sub.2-C.sub.5 alkenyl, substituted
C.sub.2-C.sub.5 alkenyl, aryl, substituted aryl, cycloalkyl,
substituted cycloalkyl, heteroaryl, substituted heteroaryl,
heterocyclic, substituted heterocyclic; R.sup.2 is hydrogen or
C.sub.1-C.sub.4 alkyl; or salts, tautomers and/or solvates thereof,
wherein the conjugate is subsequently absorbed by cancer cells and
wherein said conjugate targets said cancer cells at a rate of at
least 2:1 greater than it targets non-cancerous cells.
4. The conjugate of claim 3, wherein Y is >NR.sup.1.
5. The conjugate of claim 4, wherein L is a linker of the formula
--NH--R--NH-- where R is selected from the group consisting
-(oxyalkylene).sub.n- (where n is 1 to 10), alkylene, alkarylene,
arylalkylene, arylene, heteroarylene, heterocycloalkylene,
alkenylene, alkynylene, and cycloalkylene, each optionally
substituted with 1 to 5 substituents selected from the group
consisting of alkoxy, substituted alkoxy, amino, substituted amino,
acyl, carboxyl, carboxyl esters, cyano, halo, hydroxyl, and
thiol.
6. The conjugate of claim 3 wherein, X is a pro-fluorescent
fluorescein moiety.
7. A conjugate of the formula HA and IIB: ##STR00089## X' is a
pro-fluorescent moiety; Y' is >NR.sup.1 where R.sup.1 is
hydrogen or C.sub.1-C.sub.4 alkyl; R.sup.2 is hydrogen or
C.sub.1-C.sub.4 alkyl; L' is a linker of the formula
--NH--R.sup.3--NH-- where R.sup.3 is selected from the group
consisting -(oxyalkylene).sub.n-, alkylene, alkarylene, and
arylalkylene, or salts, tautomers and/or solvates thereof, wherein
the conjugate is subsequently absorbed by cancer cells and wherein
said conjugate targets said cancer cells at a rate of at least 2:1
greater than it targets non-cancerous cells.
8. The conjugate of claim 7, wherein X' is a fluorescein
diester.
9. The conjugate of claim 8, wherein each ester is independently of
the formula --C(O)R.sup.2 where R.sup.2 is a C.sub.2-C.sub.20 alkyl
group optionally containing 1 to 6 heteroatoms selected from --O--,
--S--, carbonyl, >NR.sup.1, --NR.sup.1R.sup.1, --OH, --SH,
phosphate and sulfate.
10. A composition comprising an inert carrier and a diagnositically
effective amount of a conjugate of claim 1.
11. A composition comprising an inert carrier and a diagnositically
effective amount of a conjugate of claim 3.
12. A composition comprising an inert carrier and a diagnositically
effective amount of a conjugate of claim 7.
13. A conjugate of the formula including salts, solvates and
tautomers thereof: ##STR00090## where: R.sup.50 and R.sup.51 are
independently C.sub.2-C.sub.18 alkyl or cycloalkyl groups either or
both optionally containing 1 to 8 heteroatoms selected from the
group consisting of oxygen, S(O).sub.x, >NR.sup.53,
--OP(O).sub.yH, --OS(O).sub.zH, --C(O)--, amino, C.sub.1-C.sub.6
alkylamino, di(C.sub.1-C.sub.6 dialkyl)amino, --C(O)O--,
--C(O)NR.sup.13--, --C(O)OH, --OH, and oxo wherein y and z are
independently 1 or 2, and x is 0, 1 or 2; R.sup.53 is hydrogen or
C.sub.1-C.sub.6 alkyl; R.sup.52 is selected from hydrogen and
C.sub.1-C.sub.4 alkyl; L.sup.a is a linker of from 1 to 20 carbon
atoms and 1 to 6 heteroatoms; selected from the group consisting of
oxygen, S(O).sub.x, >NR.sup.53, --OP(O).sub.yH, --OS(O).sub.zH,
--C(O)--, amino, C.sub.1-C.sub.6 alkylamino, di(C.sub.1-C.sub.6
dialkyl)amino, --C(O)O--, --C(O)NR.sup.3--, --C(O)OH, --OH, and oxo
wherein y and z are independently 1 or 2, and x is 0, 1 or 2; and
or pharmaceutically acceptable salts and/or solvates thereof,
wherein the conjugate is subsequently absorbed by cancer cells and
wherein said conjugate targets said cancer cells at a rate of at
least 2:1 greater than it targets non-cancerous cells.
14. A conjugate of the formula including salts, solvates and
tautomers thereof: ##STR00091## where: R.sup.50 and R.sup.51 are
independently C.sub.2-C.sub.18 alkyl or cycloalkyl groups either or
both optionally containing 1 to 8 heteroatoms selected from the
group consisting of oxygen, S(O).sub.x, >NR.sup.53,
--OP(O).sub.yH, --OS(O).sub.zH, --C(O)--, amino, C.sub.1-C.sub.6
alkylamino, di(C.sub.1-C.sub.6 dialkyl)amino, --C(O)O--,
--C(O)NR.sup.103--, --C(O)OH, --OH, and oxo wherein y and z are
independently 1 or 2, and x is 0, 1 or 2; R.sup.53 is hydrogen or
C.sub.1-C.sub.6 alkyl; R.sup.52 is selected from hydrogen and
C.sub.1-C.sub.4 alkyl; L.sup.a is a linker of from 1 to 20 carbon
atoms and 1 to 6 heteroatoms; selected from the group consisting of
oxygen, S(O).sub.x, >NR.sup.53, --OP(O).sub.yH, --OS(O).sub.zH,
--C(O)--, amino, C.sub.1-C.sub.6 alkylamino, di(C.sub.1-C.sub.6
dialkyl)amino, --C(O)O--, --C(O)NR.sup.3--, --C(O)OH, --OH, and oxo
wherein y and z are independently 1 or 2, and x is 0, 1 or 2; and
or pharmaceutically acceptable salts and/or solvates thereof,
wherein the conjugate is subsequently absorbed by cancer cells and
wherein said conjugate targets said cancer cells at a rate of at
least 2:1 greater than it targets non-cancerous cells.
15. A conjugate according to claim 3 of the formula including
salts, solvates and tautomers thereof: TABLE-US-00008 ##STR00092##
where L.sup.1 Y (from Y to X) NH
--(CH.sub.2CH.sub.2O).sub.3CH.sub.2CH.sub.2NHCH.sub.2C(O)NH-- NH
--(CH.sub.2CH.sub.2O).sub.3CH.sub.2CH.sub.2NHC(S)NH-- NH
--CH.sub.2CH.sub.2-- O
--(CH.sub.2CH.sub.2O).sub.3CH.sub.2CH.sub.2OC(S)NH-- NH
--(CH.sub.2CH.sub.2O).sub.3CH.sub.2CH.sub.2OC(S)NH-- NH
--CH.sub.2CH.sub.2-- NH
--(CH.sub.2CH.sub.2O).sub.3CH.sub.2CH.sub.2NHCH.sub.2C(O)NH-- NH
--(CH.sub.2CH.sub.2O).sub.3CH.sub.2CH.sub.2NHC(S)NH-- NH
--CH.sub.2CH.sub.2-- O
--(CH.sub.2CH.sub.2O).sub.3CH.sub.2CH.sub.2OC(S)NH-- NH
--(CH.sub.2CH.sub.2O).sub.3CH.sub.2CH.sub.2OC(S)NH-- NH
--(CH.sub.2).sub.5-- X ##STR00093## ##STR00094## ##STR00095##
##STR00096## ##STR00097## ##STR00098## ##STR00099## ##STR00100##
##STR00101## ##STR00102## ##STR00103## ##STR00104## Imaging Agent
di(methoxyethoxy)carbonylethyl carbonyloxy)5-fluorescein
di(methoxyethoxy)carbonylethyl carbonyloxy)fluorescein
di(methoxyethoxy)carbonylethyl carbonyloxy)5-fluorescein
di-(n-butylcarbonyloxy)5-fluorescein
di-(n-hexacarbonyloxy)5-fluorescein
di-(8-carboxyhexacarbonyloxy)5-fluorescein
di(methoxyethoxy)carbonylethyl carbonyloxy)6-fluorescein
di(methoxyethoxy)carbonylethyl carbonyloxy)6-fluorescein
di(methoxyethoxy)carbonylethyl carbonyloxy)6-fluorescein
5',7'-dichloro-di-(8-carboxyhexacarbonyloxy)5-fluorescein
di(methoxyethoxy)carbonylethyl carbonyloxy)5-fluorescein
wherein the conjugate is subsequently absorbed by cancer cells and
wherein said conjugate targets said cancer cells at a rate of at
least 2:1 greater than it targets non-cancerous cells.
16. A conjugate according to claim 3 of the formula including
salts, solvates and tautomers thereof: TABLE-US-00009 ##STR00105##
where L.sup.1 Y (from Y to X) NH
--(CH.sub.2CH.sub.2O).sub.3CH.sub.2CH.sub.2NHCH.sub.2C(O)NH-- NH
--(CH.sub.2CH.sub.2O).sub.3CH.sub.2CH.sub.2NHC(S)NH-- NH
--CH.sub.2CH.sub.2-- O
--(CH.sub.2CH.sub.2O).sub.3CH.sub.2CH.sub.2OC(S)NH-- NH
--(CH.sub.2CH.sub.2O).sub.3CH.sub.2CH.sub.2OC(S)NH-- NH
--CH.sub.2CH.sub.2-- NH
--(CH.sub.2CH.sub.2O).sub.3CH.sub.2CH.sub.2NHCH.sub.2C(O)NH-- NH
--(CH.sub.2CH.sub.2O).sub.3CH.sub.2CH.sub.2NHC(S)NH-- NH
--CH.sub.2CH.sub.2-- O
--(CH.sub.2CH.sub.2O).sub.3CH.sub.2CH.sub.2OC(S)NH-- NH
--(CH.sub.2CH.sub.2O).sub.3CH.sub.2CH.sub.2OC(S)NH-- X ##STR00106##
##STR00107## ##STR00108## ##STR00109## ##STR00110## ##STR00111##
##STR00112## ##STR00113## ##STR00114## ##STR00115## ##STR00116##
Imaging Agent di(methoxyethoxy)carbonylethyl
carbonyloxy)5-fluorescein di(methoxyethoxy)carbonylethyl
carbonyloxy)fluorescein di(methoxyethoxy)carbonylethyl
carbonyloxy)5-fluorescein di-(n-butylcarbonyloxy)5-fluorescein
di-(n-hexacarbonyloxy)5-fluorescein
di-(8-carboxyhexacarbonyloxy)5-fluorescein
di(methoxyethoxy)carbonylethyl carbonyloxy)6-fluorescein
di(methoxyethoxy)carbonylethyl carbonyloxy)6-fluorescein
di(methoxyethoxy)carbonylethyl carbonyloxy)6-fluorescein
5',7'-dichloro-di-(8-carboxyhexacarbonyloxy)5-fluorescein
wherein the conjugate is subsequently absorbed by cancer cells and
wherein said conjugate targets said cancer cells at a rate of at
least 2:1 greater than it targets non-cancerous cells.
17. A conjugate according to claim 13 of the formula including
salts, solvates and tautomers thereof: TABLE-US-00010 ##STR00117##
where R.sup.50/R.sup.51 R.sup.2 L.sup.a
CH.sub.3OCH.sub.2CH.sub.2OC(O)CH.sub.2CH.sub.2C(O)O--/
CH.sub.3OCH.sub.2CH.sub.2OC(O)CH.sub.2CH.sub.2C(O)O-- ##STR00118##
CH.sub.3OCH.sub.2CH.sub.2OC(O)CH.sub.2CH.sub.2C(O)O--/
CH.sub.3OCH.sub.2CH.sub.2OC(O)CH.sub.2CH.sub.2C(O)O-- H.sub.3
##STR00119## CH.sub.3OCH.sub.2CH.sub.2OC(O)CH.sub.2CH.sub.2C(O)O--/
--NHCH.sub.2CH.sub.2CH.sub.2(OCH.sub.2CH.sub.2).sub.3CH.sub.3NHC(.dbd.S)N-
H-- CH.sub.3OCH.sub.2CH.sub.2OC(O)CH.sub.2CH.sub.2C(O)O--
CH.sub.3OCH.sub.2CH.sub.2OC(O)CH.sub.2CH.sub.2C(O)O--/
CH.sub.3OCH.sub.2CH.sub.2OC(O)CH.sub.2CH.sub.2C(O)O-- ##STR00120##
CH.sub.3OCH.sub.2CH.sub.2OC(O)CH.sub.2CH.sub.2C(O)O--/
CH.sub.3OCH.sub.2CH.sub.2OC(O)CH.sub.2CH.sub.2C(O)O-- ##STR00121##
CH.sub.3OCH.sub.2CH.sub.2OC(O)CH.sub.2CH.sub.2C(O)O--/
CH.sub.3CH.sub.2C(O)O-- ##STR00122##
CH.sub.3O.sub.2C(CH.sub.2CH.sub.2O).sub.3C(O)(CH.sub.2CH.sub.2O).sub.3C(O)-
O--
CH.sub.3O.sub.2C(CH.sub.2CH.sub.2O).sub.3C(O)(CH.sub.2CH.sub.2O).sub.3-
C(O)O-- ##STR00123##
CH.sub.3O(CH.sub.2CH.sub.2O).sub.2CH.sub.2C(O)O--/
CH.sub.3O(CH.sub.2CH.sub.2O).sub.2CH.sub.2C(O)O-- ##STR00124##
wherein the conjugate is subsequently absorbed by cancer cells and
wherein said conjugate targets said cancer cells at a rate of at
least 2:1 greater than it targets non-cancerous cells.
18. A conjugate according to claim 14 of the formula including
salts, solvates and tautomers thereof: TABLE-US-00011 ##STR00125##
where R.sup.50/R.sup.51 L.sup.a
CH.sub.3OCH.sub.2CH.sub.2OC(O)CH.sub.2CH.sub.2C(O)O--/
CH.sub.3OCH.sub.2CH.sub.2OC(O)CH.sub.2CH.sub.2C(O)O-- ##STR00126##
CH.sub.3OCH.sub.2CH.sub.2OC(O)CH.sub.2CH.sub.2C(O)O--/
CH.sub.3OCH.sub.2CH.sub.2OC(O)CH.sub.2CH.sub.2C(O)O-- ##STR00127##
CH.sub.3OCH.sub.2CH.sub.2OC(O)CH.sub.2CH.sub.2C(O)O--/
CH.sub.3OCH.sub.2CH.sub.2OC(O)CH.sub.2CH.sub.2C(O)O--
--NHCH.sub.2CH.sub.2CH.sub.2(OCH.sub.2CH.sub.2).sub.3CH.sub.2NHC(.dbd.S)N-
H-- CH.sub.3OCH.sub.2CH.sub.2OC(O)CH.sub.2CH.sub.2C(O)O--/
CH.sub.3OCH.sub.2CH.sub.2OC(O)CH.sub.2CH.sub.2C(O)O-- ##STR00128##
CH.sub.3OCH.sub.2CH.sub.2OC(O)CH.sub.2CH.sub.2C(O)O--
CH.sub.3OCH.sub.2CH.sub.2OC(O)CH.sub.2CH.sub.2C(O)O-- ##STR00129##
CH.sub.3OCH.sub.2CH.sub.2OC(O)CH.sub.2CH.sub.2C(O)O--/
CH.sub.3CH.sub.2C(O)O-- ##STR00130##
CH.sub.3O.sub.2C(CH.sub.2CH.sub.2O).sub.3C(O)(CH.sub.2CH.sub.2O).sub.3C(O)-
O--
CH.sub.3O.sub.2C(CH.sub.2CH.sub.2O).sub.3C(O)(CH.sub.2CH.sub.2O).sub.3-
C(O)O-- ##STR00131##
CH.sub.3O(CH.sub.2CH.sub.2O).sub.2CH.sub.2C(O)O--/
CH.sub.3O(CH.sub.2CH.sub.2O).sub.2CH.sub.2C(O)O-- ##STR00132##
where the conjugate is subsequently absorbed by cancer cells and
wherein said conjugate targets said cancer cells at a rate of at
least 2:1 greater than it targets non-cancerous cells.
19. A composition comprising an acceptable inert aqueous carrier
and a diagnositically effective amount of a conjugate of claim
1.
20. The composition of claim 19, wherein said composition comprises
water and optionally a water soluble or miscible co-solvent.
21. The composition of claim 20, wherein said co-solvent is
selected from the group consisting of DMSO, ethanol, and
octanol.
22. The composition of claim 21, wherein the composition comprises
water and DMSO at a range of from 1:100 to 100:1.
23. The composition of claim 22, wherein the composition comprises
water and DMSO at a range of about 9:1.
24. A method for detecting cancer cells in a cellular mass
suspected of containing cancer cells which method comprises:
contacting said mass with a composition comprising an inert aqueous
carrier and a conjugate of claim 1 and applying said composition to
a cellular composition suspected of containing cancer cells;
maintaining said contact for a sufficient period of time to permit
absorption of said conjugate into said cancer cells with
specificity if such cancer cells are present and subsequent
intracellular demasking of said masked fluorescent imaging agent;
and assessing the presence of intracellular fluorescence in said
cellular mass by application of UV light comprising a wavelength
that will excite said fluorescent imaging agent as an indicia of
the presence of cancer cells in said mass.
25. The composition of claim 24, wherein said composition comprises
water and optionally a water soluble or miscible co-solvent.
26. The composition of claim 25, wherein said co-solvent is
selected from the group consisting of DMSO, ethanol, and
octanol.
27. The composition of claim 26, wherein the composition comprises
water and DMSO at a range of from 1:100 to 100:1.
28. The composition of claim 27, wherein the composition comprises
water and DMSO at a range of about 9:1.
29. A method for preparing a diagnostic composition comprising a
conjugate of claim 7 which method comprises dissolving said
conjugate in DMSO and then combining said DMSO solution with water
such that the resulting composition has a ratio of about 9:1 water
to DMSO.
30. A conjugate comprising a targeting moiety wherein said
conjugate comprises the formula T-[PF].sub.n where T is a targeting
moiety capable of specifically binding to an epitope on a cancer
cell, PF is a pro-fluorescent fluorescein moiety covalently bound
to the targeting moiety wherein said pro-fluorescent moiety can be
converted to a fluorescent moiety; and n is an integer of from 1 to
100 and preferably 10 to 90.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the priority benefit of United
States provisional application nos. 62/370,130 filed on Aug. 2,
2016, 62/378,128 filed on Aug. 22, 2016, and 62/393,524 filed on
Sep. 12, 2016, all incorporated in their entirety herein by
reference. This application is also a continuation in part of U.S.
application Ser. No. 15/595,930 filed on May 15, 2017, a
continuation in part of United States application of Ser. No.
15/595,920 filed on May 15, 2017, and a continuation in part of
international application no. PCT/US2017/13531 filed Jan. 13, 2017,
all applications incorporated in their entirety herein by
reference.
1. FIELD OF THE INVENTION
[0002] This invention is directed to folic acid and pteroic acid
conjugates and methods for using such conjugates. The conjugates
described herein comprise a targeting agent that targets cancer
cells with specificity. The targeting agent is covalently bound to
a masked imaging agent through a bond or via a linker. As such, the
conjugate first targets and is then absorbed by cancer cells. Upon
absorption, the masked imaging agent is unmasked intracellularly to
permit the imaging agent to be capable of producing a detectible
signal.
2. STATE OF THE ART
[0003] The art has long recognized the value of using targeting
agents bound to imaging agents that could image aberrant mammalian
cells such as those found in solid mass tumors. One example of such
conjugates is found in U.S. Pat. No. 8,043,603. However, in that
case, the imaging agent is never masked and is fully capable of
producing a signal whether found intracellularly or
extracellularly.
[0004] Such prior art imaging agents require intravenous injection
hours prior to solid mass tumor resection so that these agents can
preferentially accumulate into the tumor. While such accumulation
provides images of such tumors, the ability to detect remnant tumor
cells remaining at the margins after surgery remains elusive as
most of the imaging agent used resides in the bulk of the tumor.
However, it is well known that remnant tumor cells are responsible,
as least in part, for recurrence of the cancer.
[0005] The use of masked imaging agents that can bind to cancer
cells with specificity and rapidly produce a signal when absorbed
by such cancer cells and become unmasked is a long felt need in the
art and one that would significantly improve remnant cancer cell
detection and subsequent removal.
3. SUMMARY OF THE INVENTION
[0006] This invention provides for compounds (conjugates),
compositions and methods whereby the compounds comprise a targeting
(or delivering) agent that targets cancer cells with specificity
and is subsequently absorbed by these cells. The targeting agent is
covalently bound to a masked fluorescent imaging agent optionally
through a linker. Upon absorption of the conjugate, the masked
fluorescent imaging agent is unmasked so that the imaging agent is
capable of producing a detectible fluorescent signal. The
conjugates are preferably included in a composition that is capable
of being applied a cellular mass suspected of containing cancer
cells such as the surface of the surgical field after resection of
the tumor.
[0007] The data establishes that the conjugates of this invention
readily target and then are absorbed by cancer cells whereupon
demasking of the imaging agent rapidly occurs thereby allowing the
imaging agent to generate a fluorescent signal. In contrast
thereto, the conjugates that remain masked do not produce a
detectible signal and thus allow the clinician to readily identify
remnant cancer cells after surgical resection of the solid mass
tumor.
[0008] In one embodiment, this invention provides for a conjugate
that comprises:
[0009] a targeting agent that targets the conjugate to cancer cells
and where the conjugate is subsequently absorbed by said cancer
cells wherein said conjugate targets said cancer cells at a rate of
at least 2:1 greater than it targets non-cancerous cells; and
[0010] a masked fluorescent imaging agent wherein the masking group
is covalently attached to the imaging agent provided that the
imaging agent is not a fluorescent-fluorescent quencher pair and
wherein the targeting agent is covalently bound to a masked
fluorescent imaging agent via a bond or a linker; and
[0011] further wherein, upon absorption of the conjugate by the
cancer cell, the masked fluorescent imaging agent is unmasked to
permit the imaging agent to be capable of producing a detectible
fluorescent signal,
[0012] preferably the targeting agent is not albumin, an antibody,
or hyaluronic acid.
[0013] In an embodiment of the invention the conjugate is not:
##STR00001##
[0014] wherein X'' is selected from the group consisting of --O--
and >NR.sup.20, L'' is a bond or linking group having 1 to 30
atoms, R'' is an alkyl group or a substituted alkyl group having
from 4 to 30 carbon atoms and optionally 1 to 6 heteroatoms
selected from oxygen, hydroxyl, sulfur, carbonyl, thiocarbonyl,
chloro, bromo, iodo, and cyano; and R.sup.20 is hydrogen or alkyl;
or
##STR00002##
where R.sup.11 and R.sup.10 are C.sub.1-C.sub.30 ester groups;
or
##STR00003##
where R' is an alkyl group or a substituted alkyl group having from
4 to 30 carbon atoms and optionally 1 to 6 heteroatoms selected
from oxygen, hydroxyl, sulfur, thiol, carbonyl, thiocarbonyl,
chloro, bromo, iodo, and cyano; or
##STR00004##
[0015] wherein R.sup.40 is C.sub.1-C.sub.6 alkyl or heteroalkyl
optionally substituted with a water solubility imparting functional
group selected from the group consisting of hydroxy, methoxy,
amino, carboxylic acid or carboxylate;
[0016] R.sup.45 is hydrogen or an optionally substituted alkyl
group;
[0017] L'' is linker comprises a linear, branched and/or cyclic
carbon chain having 1 to 20 carbon atoms and optionally comprising
unsaturated covalent bonds and heteroatoms, such as nitrogen,
oxygen, sulfur, phosphate, and/or halogens and further optionally
containing a hydrocarbon chain comprising substituted or
unsubstituted aryls, acyls, esters, alkoxyls, alkyls, carbonyls,
and hydroxyl;
[0018] folate is a folic acid moiety joined with the rest of the
conjugate via an ester of the folic acid carboxyl group; and
[0019] folamide is a folic acid moiety joined with the rest of the
conjugate via an amide of the folic acid carboxyl group.
[0020] In one embodiment, the linker comprises one or more oxygen
atoms to improve the water solubility of the conjugate optionally
in addition to any nitrogen and/or sulfur atoms. Such oxygen atoms
can take the form of hydroxyl, carbonyl, carboxyl, oxyalkylene, and
related structures. Preferably, the number of oxygen atoms is no
more than 10. More preferably, the oxygen containing linker
comprises a polyoxyalkylene group of from 1 to 10 oxyalkylene
units.
[0021] In one embodiment, the targeting agent is folic acid,
pteroic acid, folic acid alpha C.sub.1-C.sub.4 ester. The
structures of folic acid and pteroic acid are provided as
follows:
##STR00005##
[0022] As shown above, the carboxyl group proximate to the amide
bond is referred to as the alpha carboxyl group; whereas the
carboxyl group distal to the amide bond is referred to as the gamma
carboxyl group. These two carboxyl groups together with the amino
group of the amide bond form a glutamic acid residue. Without being
limited by any theory, this residue does not appear essential to
recognition of folic acid to the folate receptors on cancer cells
and, as such, allows for conjugation with or without the glutamic
acid residue. The structure of folic acid without the glutamic acid
residue provides for pteroic acid.
[0023] In one embodiment, folic acid and pteroic acid are suitable
scaffolds for the delivery of masked fluorescent imaging agents to
mammalian cancer cells that overexpress folate receptors provided
that the masking agents are bound to said imaging agents through a
covalent bond stable in the extracellular domain but capable of
unmasking when absorbed by such cancer cells. Many mammalian
cancers are well known to overexpress folate receptors including
breast cancer, ovarian cancer, cervical cancer, renal cancer, lung
cancer, brain cancer, colorectal cancer, epithelial cancer, and the
like. As such, the conjugates of this invention "target" these
cancer cells. Upon targeting, the conjugates are absorbed by these
cells and then are unmasked. Without being limited to any theory,
it is believed that intracellular enzymes such as esterases and
lipases unmask the imaging agent thereby permitting the imaging
agent to generate a detectible signal.
[0024] Accordingly, in one embodiment, there is provided a
conjugate of the formula IA and IB:
##STR00006##
[0025] where L.sup.1 is a bond or a linker,
[0026] X is a masked imaging agent, and
[0027] Y is --O-- or >NR.sup.1 where R.sup.1 is hydrogen,
C.sub.1-C.sub.4 alkyl, substituted C.sub.1-C.sub.4 alkyl;
C.sub.2-C.sub.5 alkenyl, substituted C.sub.2-C.sub.5 alkenyl, aryl,
substituted aryl, cycloalkyl, substituted cycloalkyl, heteroaryl,
substituted heteroaryl, heterocyclic, substituted heterocyclic;
[0028] R.sup.2 is hydrogen or C.sub.1-C.sub.4 alkyl;
[0029] or salts, tautomers and/or solvates thereof, wherein the
conjugate is subsequently absorbed by cancer cells and wherein said
conjugate targets said cancer cells at a rate of at least 2:1
greater than it targets non-cancerous cells.
[0030] In an embodiment of the invention the conjugate is not
##STR00007##
[0031] wherein X'' is selected from the group consisting of --O--
and >NR.sup.20, L'' is a bond or linking group having 1 to 30
atoms, R'' is an alkyl group or a substituted alkyl group having
from 4 to 30 carbon atoms and optionally 1 to 6 heteroatoms
selected from oxygen, hydroxyl, sulfur, carbonyl, thiocarbonyl,
chloro, bromo, iodo, and cyano; and R.sup.20 is hydrogen or alkyl;
or
##STR00008##
where R.sup.11 and R.sup.10 are C.sub.1-C.sub.30 ester groups;
or
##STR00009##
where R is an alkyl group or a substituted alkyl group having from
4 to 30 carbon atoms and optionally 1 to 6 heteroatoms selected
from oxygen, hydroxyl, sulfur, thiol, carbonyl, thiocarbonyl,
chloro, bromo, iodo, and cyano; or
##STR00010##
[0032] wherein R.sup.40 is C.sub.1-C.sub.6 alkyl or heteroalkyl
optionally substituted with a water solubility imparting functional
group selected from the group consisting of hydroxy, methoxy,
amino, carboxylic acid, and carboxylate;
[0033] R.sup.45 is hydrogen or an optionally substituted alkyl
group;
[0034] L'' is a bond or a linking group having 1 to 30 atoms
including --NHC(O)-- alkylene-, --NHC(O)NH-alkylene-,
--NHC(S)NH-alkylene-, and --OC(O)NH--;
[0035] folate is a folic acid moiety joined with the rest of the
conjugate via an ester of the folic acid carboxyl group; and
[0036] folamide is a folic acid moiety joined with the rest of the
conjugate via an amide of the folic acid carboxyl group.
[0037] In one embodiment, Y is >NR.sup.1.
[0038] In one embodiment, L.sup.1 is a linker of the formula
--NH--R--NH-- where R is selected from the group consisting
-(oxyalkylene).sub.n- (where n is 1 to 10), alkylene, alkarylene,
arylalkylene, arylene, heteroarylene, heterocycloalkylene,
alkenylene, alkynylene, and cycloalkylene, each optionally
substituted with 1 to 5 substituents selected from the group
consisting of alkoxy, substituted alkoxy, amino, substituted amino,
acyl, carboxyl, carboxyl esters, cyano, halo, hydroxyl, thiol, and
the like, with the proviso set forth above.
[0039] In one embodiment, X is a pro-fluorescent moiety. In a
preferred embodiment, X is a pro-fluorescent fluorescein
moiety.
[0040] In one of embodiment, there is provided a conjugate of the
formula IIA and IIB:
##STR00011##
[0041] X' is a pro-fluorescent moiety;
[0042] Y' is >NR.sup.1 where R.sup.1 is hydrogen or
C.sub.1-C.sub.4 alkyl;
[0043] R.sup.2 is hydrogen or C.sub.1-C.sub.4 alkyl;
[0044] L.sup.2 is a linker of the formula --NH--R.sup.3--NH-- where
R.sup.3 is selected from the group consisting
-(oxyalkylene).sub.n-, alkylene, alkarylene, and arylalkylene, or
salts, tautomers and/or solvates thereof, wherein the conjugate is
subsequently absorbed by cancer cells and wherein the conjugate
targets the cancer cells at a rate of at least 2:1 greater than it
targets non-cancerous cells.
[0045] In an embodiment of the invention the conjugate is not
##STR00012##
[0046] wherein X'' is selected from the group consisting of --O--
and >NR.sup.20, L'' is a bond or linking group having 1 to 30
atoms, R'' is an alkyl group or a substituted alkyl group having
from 4 to 30 carbon atoms and optionally 1 to 6 heteroatoms
selected from oxygen, hydroxyl, sulfur, carbonyl, thiocarbonyl,
chloro, bromo, iodo, and cyano; and R.sup.20 is hydrogen or alkyl;
or
##STR00013##
where R.sup.11 and R.sup.10 are C.sub.1-C.sub.30 ester groups;
or
##STR00014##
where R is an alkyl group or a substituted alkyl group having from
4 to 30 carbon atoms and optionally 1 to 6 heteroatoms selected
from oxygen, hydroxyl, sulfur, thiol, carbonyl, thiocarbonyl,
chloro, bromo, iodo, and cyano; or
##STR00015##
[0047] wherein R.sup.40 is C.sub.1-C.sub.6 alkyl or heteroalkyl
optionally substituted with a water solubility imparting functional
group selected from the group consisting of hydroxy, methoxy,
amino, carboxylic acid, and carboxylate;
[0048] R.sup.45 is hydrogen or an optionally substituted alkyl
group;
[0049] L'' is a bond or a linking group having 1 to 30 atoms
including --NHC(O)-- alkylene-, --NHC(O)NH-alkylene-,
--NHC(S)NH-alkylene-, and --OC(O)NH--;
[0050] folate is a folic acid moiety joined with the rest of the
conjugate via an ester of the folic acid carboxyl group; and
[0051] folamide is a folic acid moiety joined with the rest of the
conjugate via an amide of the folic acid carboxyl group.
[0052] In one embodiment, X' is a fluorescein diester. In another
embodiment, each ester is independently of the formula
--C(O)R.sup.2' where R.sup.2' is an C.sub.2-C.sub.20 alkyl group
optionally containing 1 to 6 heteroatoms selected from --O--,
--S--, carbonyl, >NR.sup.1, --NR.sup.1R.sup.1, --OH, --SH,
phosphate and sulfate where R.sup.1 is as defined above.
[0053] In one of its method aspects, there is provided a method for
targeting an imaging agent to folic acid expressing cancer cells
which method comprises:
[0054] a) confirming that the cancer cells express folic acid
receptors;
[0055] b) selecting a conjugate of formula IA or 18:
##STR00016##
where R.sup.2, L, X and Y are as defined above and with the
provisos provided above;
[0056] c) contacting said cancer cells with a diagnostically
effective amount of a conjugate formula IA or IB such that said
conjugate is absorbed by said cells; and
[0057] d) maintaining said cells under conditions wherein said
imaging agent is demasked under intracellular conditions.
[0058] Representative conjugates of this invention are provided in
Tables 1 and 2 below. Such conjugates include salts, tautomers
and/or solvates thereof.
TABLE-US-00001 TABLE 1 Folic Acid/Imaging Agents ##STR00017## L Y
(from Y to X) 1 NH
--(CH.sub.2CH.sub.2O).sub.3CH.sub.2CH.sub.2NHCH.sub.2C(O)NH-- 2 NH
--(CH.sub.2CH.sub.2O).sub.3CH.sub.2CH.sub.2NHC(S)NH-- 3 NH
--CH.sub.2CH.sub.2-- 4 O
--(CH.sub.2CH.sub.2O).sub.3CH.sub.2CH.sub.2OC(S)NH-- 5 NH
--(CH.sub.2CH.sub.2O).sub.3CH.sub.2CH.sub.2OC(S)NH-- 6 NH
--CH.sub.2CH.sub.2-- 7 NH
--(CH.sub.2CH.sub.2O).sub.3CH.sub.2CH.sub.2NHCH.sub.2C(O)NH-- 8 NH
--(CH.sub.2CH.sub.2O).sub.3CH.sub.2CH.sub.2NHC(S)NH-- 9 NH
--CH.sub.2CH.sub.2-- 10 O
--(CH.sub.2CH.sub.2O).sub.3CH.sub.2CH.sub.2OC(S)NH-- 11 NH
--(CH.sub.2CH.sub.2O).sub.3CH.sub.2CH.sub.2OC(S)NH-- 12 NH
--(CH.sub.2).sub.5-- X 1 ##STR00018## 2 ##STR00019## 3 ##STR00020##
4 ##STR00021## 5 ##STR00022## 6 ##STR00023## 7 ##STR00024## 8
##STR00025## 9 ##STR00026## 10 ##STR00027## 11 ##STR00028## 12
##STR00029## Imaging Agent 1 di(methoxyethoxy)carbonylethyl
carbonyloxy)5-fluorescein 2 di(methoxyethoxy)carbonylethyl
carbonyloxy)fluorescein 3 di(methoxyethoxy)carbonylethyl
carbonyloxy)5-fluorescein 4 di-(n-butylcarbonyloxy)5-fluorescein 5
di-(n-hexacarbonyloxy)5-fluorescein 6
di-(8-carboxyhexacarbonyloxy)5-fluorescein 7
di(methoxyethoxy)carbonylethyl carbonyloxy)6-fluorescein 8
di(methoxyethoxy)carbonylethyl carbonyloxy)6-fluorescein 9
di(methoxyethoxy)carbonylethyl carbonyloxy)6-fluorescein 10 11
5',7'-dichloro-di-(8-carboxyhexacarbonyloxy)5-fluorescein 12
di(methoxyethoxy)carbonylethyl carbonyloxy)5-fluorescein
[0059] wherein the conjugate is subsequently absorbed by cancer
cells and wherein the conjugate targets the cancer cells at a rate
of at least 2:1 greater than it targets non-cancerous cells. In an
embodiment of this invention the conjugate is not
##STR00030##
[0060] wherein X'' is selected from the group consisting of --O--
and >NR.sup.20, L'' is a bond or linking group having 1 to 30
atoms, R'' is an alkyl group or a substituted alkyl group having
from 4 to 30 carbon atoms and optionally 1 to 6 heteroatoms
selected from oxygen, hydroxyl, sulfur, carbonyl, thiocarbonyl,
chloro, bromo, iodo, and cyano; and R.sup.20 is hydrogen or
alkyl.
TABLE-US-00002 TABLE 2 Pteroic Acid/Imaging Agents ##STR00031## L Y
(from Y to X) 13 NH
--(CH.sub.2CH.sub.2O).sub.3CH.sub.2CH.sub.2NHCH.sub.2C(O)NH-- 14 NH
--(CH.sub.2CH.sub.2O).sub.3CH.sub.2CH.sub.2NHC(S)NH-- 15 NH
--CH.sub.2CH.sub.2-- 16 O
--(CH.sub.2CH.sub.2O).sub.3CH.sub.2CH.sub.2OC(S)NH-- 17 NH
--(CH.sub.2CH.sub.2O).sub.3CH.sub.2CH.sub.2OC(S)NH-- 18 NH
--CH.sub.2CH.sub.2-- 19 NH
--(CH.sub.2CH.sub.2O).sub.3CH.sub.2CH.sub.2NHCH.sub.2C(O)NH-- 20 NH
--(CH.sub.2CH.sub.2O).sub.3CH.sub.2CH.sub.2NHC(S)NH-- 21 NH
--CH.sub.2CH.sub.2-- 22 O
--(CH.sub.2CH.sub.2O).sub.3CH.sub.2CH.sub.2OC(S)NH-- 23 NH
--(CH.sub.2CH.sub.2O).sub.3CH.sub.2CH.sub.2OC(S)NH-- X 13
##STR00032## 14 ##STR00033## 15 ##STR00034## 16 ##STR00035## 17
##STR00036## 18 ##STR00037## 19 ##STR00038## 20 ##STR00039## 21
##STR00040## 22 ##STR00041## 23 ##STR00042## Imaging Agent 13
di(methoxyethoxy)carbonylethyl carbonyloxy)5-fluorescein 14
di(methoxyethoxy)carbonylethyl carbonyloxy)fluorescein 15
di(methoxyethoxy)carbonylethyl carbonyloxy)5-fluorescein 16
di-(n-butylcarbonyloxy)5-fluorescein 17
di-(n-hexacarbonyloxy)5-fluorescein 18
di-(8-carboxyhexacarbonyloxy)5-fluorescein 19
di(methoxyethoxy)carbonylethyl carbonyloxy)6-fluorescein 20
di(methoxyethoxy)carbonylethyl carbonyloxy)6-fluorescein 21
di(methoxyethoxy)carbonylethyl carbonyloxy)6-fluorescein 22 23
5',7'-dichloro-di-(8-carboxyhexacarbonyloxy)5-fluorescein
[0061] wherein the conjugate is subsequently absorbed by cancer
cells and wherein the conjugate targets the cancer cells at a rate
of at least 2:1 greater than it targets non-cancerous cells.
[0062] In one embodiment, there is provided a conjugate of the
formula including salts, solvates and tautomers thereof:
##STR00043##
[0063] where:
[0064] R.sup.50 and R.sup.51 are independently C.sub.2-C.sub.18
alkyl or cycloalkyl groups either or both optionally containing 1
to 8 heteroatoms selected from the group consisting of oxygen,
S(O).sub.x, >NR.sup.53, --OP(O).sub.yH, --OS(O).sub.zH,
--C(O)--, amino, C.sub.1-C.sub.6 alkylamino, di(C.sub.1-C.sub.6
dialkyl)amino, --C(O)O--, --C(O)NR.sup.103--, --C(O)OH, --OH, and
oxo wherein y and z are independently 1 or 2, and x is 0, 1 or
2;
[0065] R.sup.53 is hydrogen or C.sub.1-C.sub.6 alkyl;
[0066] R.sup.52 is selected from hydrogen and C.sub.1-C.sub.4
alkyl;
[0067] L.sup.a is a linker of from 1 to 20 carbon atoms and 1 to 6
heteroatoms; selected from the group consisting of oxygen,
S(O).sub.x, >NR.sup.53, --OP(O).sub.yH, --OS(O)--.sub.zH,
--C(O)--, amino, C.sub.1-C.sub.6 alkylamino, di(C.sub.1-C.sub.6
dialkyl)amino, --C(O)O--, --C(O)NR.sup.53--, --C(O)OH, --OH, and
oxo wherein y and z are independently 1 or 2, and x is 0, 1 or 2;
and
[0068] or salts and/or solvates thereof, wherein the conjugate is
subsequently absorbed by cancer cells and wherein the conjugate
targets the cancer cells at a rate of at least 2:1 greater than it
targets non-cancerous cells.
[0069] In an embodiment of the invention the conjugate is not
##STR00044##
[0070] wherein X'' is selected from the group consisting of --O--
and >NR.sup.20, L'' is a bond or linking group having 1 to 30
atoms, R'' is an alkyl group or a substituted alkyl group having
from 4 to 30 carbon atoms and optionally 1 to 6 heteroatoms
selected from oxygen, hydroxyl, sulfur, carbonyl, thiocarbonyl,
chloro, bromo, iodo, and cyano; and R.sup.20 is hydrogen or alkyl;
or
##STR00045##
where R.sup.11 and R.sup.10 are C.sub.1-C.sub.30 ester groups;
or
##STR00046##
where R is an alkyl group or a substituted alkyl group having from
4 to 30 carbon atoms and optionally 1 to 6 heteroatoms selected
from oxygen, hydroxyl, sulfur, thiol, carbonyl, thiocarbonyl,
chloro, bromo, iodo, and cyano; or
##STR00047##
[0071] wherein R.sup.40 is C.sub.1-C.sub.6 alkyl or heteroalkyl
optionally substituted with a water solubility imparting functional
group selected from the group consisting of hydroxy, methoxy,
amino, carboxylic acid, and carboxylate;
[0072] R.sup.45 is hydrogen or an optionally substituted alkyl
group;
[0073] L'' is a bond or a linking group having 1 to 30 atoms
including --NHC(O)-- alkylene-, --NHC(O)NH-alkylene-,
--NHC(S)NH-alkylene-, and --OC(O)NH--;
[0074] folate is a folic acid moiety joined with the rest of the
conjugate via an ester of the folic acid carboxyl group; and
[0075] folamide is a folic acid moiety joined with the rest of the
conjugate via an amide of the folic acid carboxyl group.
[0076] In one embodiment there is provided a conjugate of the
formula including salts, solvates and tautomers thereof:
##STR00048##
[0077] where:
[0078] R.sup.50 and R.sup.51 are independently C.sub.2-C.sub.18
alkyl or cycloalkyl groups either or both optionally containing 1
to 8 heteroatoms selected from the group consisting of oxygen,
S(O).sub.x, >NR.sup.53, --OP(O).sub.yH, --OS(O).sub.zH,
--C(O)--, amino, C.sub.1-C.sub.6 alkylamino, di(C.sub.1-C.sub.6
dialkyl)amino, --C(O)O--, --C(O)NR.sup.103--, --C(O)OH, --OH, and
oxo wherein y and z are independently 1 or 2, and x is 0, 1 or
2;
[0079] R.sup.53 is hydrogen or C.sub.1-C.sub.6 alkyl;
[0080] R.sup.52 is selected from hydrogen and C.sub.1-C.sub.4
alkyl;
[0081] L.sup.a is a linker of from 1 to 20 carbon atoms and 1 to 6
heteroatoms; selected from the group consisting of oxygen,
S(O).sub.x, >NR.sup.53, --OP(O).sub.yH, --OS(O).sub.zH,
--C(O)--, amino, C.sub.1-C.sub.6 alkylamino, di(C.sub.1-C.sub.6
dialkyl)amino, --C(O)O--, --C(O)NR.sup.53--, --C(O)OH, --OH, and
oxo wherein y and z are independently 1 or 2, and x is 0, 1 or 2;
and
[0082] or salts and/or solvates thereof, wherein the conjugate is
subsequently absorbed by cancer cells and wherein the conjugate
targets the cancer cells at a rate of at least 2:1 greater than it
targets non-cancerous cells.
[0083] Such conjugates including salts, solvates and tautomers
thereof are provided in tables 3 and 4 below:
TABLE-US-00003 TABLE 3 ##STR00049## where R.sup.50/R.sup.51 R.sup.2
L.sup.a CH.sub.3OCH.sub.2CH.sub.2OC(O)CH.sub.2CH.sub.2C(O)O--/
CH.sub.3OCH.sub.2CH.sub.2OC(O)CH.sub.2CH.sub.2C(O)O-- H
##STR00050## CH.sub.3OCH.sub.2CH.sub.2OC(O)CH.sub.2CH.sub.2C(O)O--/
CH.sub.3OCH.sub.2CH.sub.2OC(O)CH.sub.2CH.sub.2C(O)O-- CH.sub.3
##STR00051## CH.sub.3OCH.sub.2CH.sub.2OC(O)CH.sub.2CH.sub.2C(O)O--/
H
--NHCH.sub.2CH.sub.2CH.sub.2(OCH.sub.2CH.sub.2).sub.3CH.sub.3NHC(.dbd.S)N-
H-- CH.sub.3OCH.sub.2CH.sub.2OC(O)CH.sub.2CH.sub.2C(O)O--
CH.sub.3OCH.sub.2CH.sub.2OC(O)CH.sub.2CH.sub.2C(O)O--/
CH.sub.3OCH.sub.2CH.sub.2OC(O)CH.sub.2CH.sub.2C(O)O-- H
##STR00052## CH.sub.3OCH.sub.2CH.sub.2OC(O)CH.sub.2CH.sub.2C(O)O--/
CH.sub.3OCH.sub.2CH.sub.2OC(O)CH.sub.2CH.sub.2C(O)O-- H
##STR00053## CH.sub.3OCH.sub.2CH.sub.2OC(O)CH.sub.2CH.sub.2C(O)O--/
CH.sub.3CH.sub.2C(O)O-- H ##STR00054##
CH.sub.3O.sub.2C(CH.sub.2CH.sub.2O).sub.3C(O)(CH.sub.2CH.sub.2O).sub.3C(O)-
O--
CH.sub.3O.sub.2C(CH.sub.2CH.sub.2O).sub.3C(O)(CH.sub.2CH.sub.2O).sub.3-
C(O)O-- H ##STR00055##
CH.sub.3O(CH.sub.2CH.sub.2O).sub.2CH.sub.2C(O)O--/
CH.sub.3O(CH.sub.2CH.sub.2O).sub.2CH.sub.2C(O)O-- H
##STR00056##
[0084] wherein the conjugate is subsequently absorbed by cancer
cells and wherein the conjugate targets the cancer cells at a rate
of at least 2:1 greater than it targets non-cancerous cells. In an
embodiment of this invention the conjugate is not
##STR00057##
[0085] wherein X'' is selected from the group consisting of --O--
and >NR.sup.20, L'' is a bond or linking group having 1 to 30
atoms, R'' is an alkyl group or a substituted alkyl group having
from 4 to 30 carbon atoms and optionally 1 to 6 heteroatoms
selected from oxygen, hydroxyl, sulfur, carbonyl, thiocarbonyl,
chloro, bromo, iodo, and cyano; and R.sup.20 is hydrogen or
alkyl.
TABLE-US-00004 TABLE 4 ##STR00058## where R.sup.50/R.sup.51 L.sup.a
CH.sub.3OCH.sub.2CH.sub.2OC(O)CH.sub.2CH.sub.2C(O)O--/
CH.sub.3OCH.sub.2CH.sub.2OC(O)CH.sub.2CH.sub.2C(O)O-- ##STR00059##
CH.sub.3OCH.sub.2CH.sub.2OC(O)CH.sub.2CH.sub.2C(O)O--/
CH.sub.3OCH.sub.2CH.sub.2OC(O)CH.sub.2CH.sub.2C(O)O-- ##STR00060##
CH.sub.3OCH.sub.2CH.sub.2OC(O)CH.sub.2CH.sub.2C(O)O--/
CH.sub.3OCH.sub.2CH.sub.2OC(O)CH.sub.2CH.sub.2C(O)O--
--NHCH.sub.2CH.sub.2CH.sub.2(OCH.sub.2CH.sub.2).sub.3CH.sub.2NHC(.dbd.S)N-
H-- CH.sub.3OCH.sub.2CH.sub.2OC(O)CH.sub.2CH.sub.2C(O)O--/
CH.sub.3OCH.sub.2CH.sub.2OC(O)CH.sub.2CH.sub.2C(O)O-- ##STR00061##
CH.sub.3OCH.sub.2CH.sub.2OC(O)CH.sub.2CH.sub.2C(O)O--
CH.sub.3OCH.sub.2CH.sub.2OC(O)CH.sub.2CH.sub.2C(O)O-- ##STR00062##
CH.sub.3OCH.sub.2CH.sub.2OC(O)CH.sub.2CH.sub.2C(O)O--/
CH.sub.3CH.sub.2C(O)O-- ##STR00063##
CH.sub.3O.sub.2C(CH.sub.2CH.sub.2O).sub.3C(O)(CH.sub.2CH.sub.2O).sub.3C(O)-
O--
CH.sub.3O.sub.2C(CH.sub.2CH.sub.2O).sub.3C(O)(CH.sub.2CH.sub.2O).sub.3-
C(O)O-- ##STR00064##
CH.sub.3O(CH.sub.2CH.sub.2O).sub.2CH.sub.2C(O)O--/
CH.sub.3O(CH.sub.2CH.sub.2O).sub.2CH.sub.2C(O)O-- ##STR00065##
[0086] wherein the conjugate is subsequently absorbed by cancer
cells and wherein the conjugate targets the cancer cells at a rate
of at least 2:1 greater than it targets non-cancerous cells.
4. BRIEF DESCRIPTION OF THE DRAWINGS
[0087] FIG. 1 illustrates that an ovarian cancer cell line (SKOV3
cells) become fluorescent when exposed to the conjugates of this
invention.
[0088] FIGS. 2A and 2B illustrate that the mechanism by which the
conjugates of this invention render these cancer cells fluorescent
is via the folic acid receptor on these cells.
5. DETAILED DESCRIPTION OF THE INVENTION
[0089] After reading this description it will become apparent to
one skilled in the art how to implement the invention in various
alternative embodiments and alternative applications. However, all
the various embodiments of the present invention will not be
described herein. It will be understood that the embodiments
presented here are presented by way of an example only, and not
limitation. As such, this detailed description of various
alternative embodiments should not be construed to limit the scope
or breadth of the present invention as set forth below.
[0090] Before the present invention is disclosed and described, it
is to be understood that the aspects described below are not
limited to specific compositions, methods of preparing such
compositions, or uses thereof as such may, of course, vary. It is
also to be understood that the terminology used herein is for the
purpose of describing particular aspects only and is not intended
to be limiting.
[0091] The specification is divided into various sections only for
the reader's convenience and disclosure found in any section may be
combined with that in another section. Titles or subtitles may be
used in the specification for the convenience of a reader, which
are not intended to influence the scope of the present
invention.
5. DEFINITIONS
[0092] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. In this
specification and in the claims that follow, reference will be made
to a number of terms that shall be defined to have the following
meanings:
[0093] The terminology used herein is for the purpose of describing
particular embodiments only and is not intended to be limiting of
the invention. As used herein, the singular forms "a", "an" and
"the" are intended to include the plural forms as well, unless the
context clearly indicates otherwise.
[0094] "Optional" or "optionally" means that the subsequently
described event or circumstance can or cannot occur, and that the
description includes instances where the event or circumstance
occurs and instances where it does not.
[0095] The term "about" when used before a numerical designation,
e.g., temperature, time, amount, concentration, and such other,
including a range, indicates approximations which may vary by (+)
or (-) 10%, 5%, 1%, or any subrange or subvalue there between.
Preferably, the term "about" when used with regard to a dose amount
means that the dose may vary by +/-10%.
[0096] "Comprising" or "comprises" is intended to mean that the
compositions and methods include the recited elements, but not
excluding others. "Consisting essentially of" when used to define
compositions and methods, shall mean excluding other elements of
any essential significance to the combination for the stated
purpose. Thus, a composition consisting essentially of the elements
as defined herein would not exclude other materials or steps that
do not materially affect the basic and novel characteristic(s) of
the claimed invention. "Consisting of" shall mean excluding more
than trace elements of other ingredients and substantial method
steps. Embodiments defined by each of these transition terms are
within the scope of this invention.
[0097] "Alkyl" refers to monovalent saturated aliphatic hydrocarbyl
groups having from 1 to 10 carbon atoms and preferably 1 to 6
carbon atoms. This term includes, by way of example, linear and
branched hydrocarbyl groups such as methyl (CH.sub.3--), ethyl
(CH.sub.3CH.sub.2--), n-propyl (CH.sub.3CH.sub.2CH.sub.2--),
iso-propyl ((CH.sub.3).sub.2CH--), n-butyl
(CH.sub.3CH.sub.2CH.sub.2CH.sub.2--), iso-butyl
((CH.sub.3).sub.2CHCH.sub.2--), sec-butyl
((CH.sub.3)(CH.sub.3CH.sub.2)CH--), t-butyl ((CH.sub.3).sub.3C--),
n-pentyl (CH.sub.3CH.sub.2CH.sub.2CH.sub.2CH.sub.2--), and
neo-pentyl ((CH.sub.3).sub.3CCH.sub.2--).
[0098] "Heteroalkyl" refers to an alkyl group wherein 1-5 carbon
atoms are replaced with one or more heteroatoms selected from
>NR.sup.30, --S--, --S(O)--, --S(O).sub.2--, and --O--, where
R.sup.30 is hydrogen, C.sub.3-C.sub.6 alkyl, or --C(O)R.sup.31 is
hydrogen or C.sub.1-C.sub.6 alkyl.
[0099] "Alkenyl" refers to monovalent straight or branched
hydrocarbyl groups having from 2 to 10 carbon atoms and preferably
2 to 6 carbon atoms or preferably 2 to 4 carbon atoms and having at
least 1 and preferably from 1 to 2 sites of vinyl (>C.dbd.C<)
unsaturation. Such groups are exemplified, for example, by vinyl,
allyl, and but-3-en-1-yl. Included within this term are the cis and
trans isomers or mixtures of these isomers.
[0100] "Substituted alkyl" refers to an alkyl group having from 1
to 5, preferably 1 to 3, or more preferably 1 to 2 substituents
selected from the group consisting of alkoxy, substituted alkoxy,
acyl, acylamino, acyloxy, amino, substituted amino, aminocarbonyl,
aminocarbonylamino, aminocarbonyloxy, aryl, substituted aryl,
aryloxy, substituted aryloxy, carboxyl, carboxyl ester, (carboxyl
ester)amino, (carboxyl ester)oxy, cyano, cycloalkyl, substituted
cycloalkyl, cycloalkyloxy, substituted cycloalkyloxy, halo,
hydroxy, heteroaryl, substituted heteroaryl, heteroaryloxy,
substituted heteroaryloxy, heterocyclic, substituted heterocyclic,
heterocyclyloxy, substituted heterocyclyloxy, oxo and nitro.
[0101] "Substituted alkenyl" refers to alkenyl groups having from 1
to 5 substituents, and preferably 1 to 2 substituents, as defined
above for substituted alkyl provided that any hydroxyl substitution
is not attached to a vinyl (unsaturated) carbon atom.
[0102] "Alkylene" refers to divalent saturated aliphatic
hydrocarbyl groups having from 1 to 10 carbon atoms, preferably
having from 1 to 6 and more preferably 1 to 3 carbon atoms that are
either straight-chained or branched. This term is exemplified by
groups such as methylene (--CH.sub.2--), ethylene
(--CH.sub.2CH.sub.2--), n-propylene (--CH.sub.2CH.sub.2CH.sub.2--),
iso-propylene (--CH.sub.2CH(CH.sub.3)-- or
--CH(CH.sub.3)CH.sub.2--), butylene
(--CH.sub.2CH.sub.2CH.sub.2CH.sub.2--), iso-butylene
(--CH.sub.2CH(CH.sub.3)CH.sub.2--), sec-butylene
(--CH.sub.2CH.sub.2(CH.sub.3)CH--), and the like.
[0103] "Substituted alkylene" refers to an alkylene group having
from from 1 to 5, preferably 1 to 3, or more preferably 1 to 2
substituents as defined above for substituted alkyl.
[0104] "Alkoxy" refers to the group --O-alkyl wherein alkyl is
defined herein. Alkoxy includes, by way of example, methoxy,
ethoxy, n-propoxy, iso-propoxy, n-butoxy, t-butoxy, sec-butoxy, and
n-pentoxy.
[0105] "Substituted alkoxy" refers to the group --O-(substituted
alkyl) wherein substituted alkyl is defined herein.
[0106] "Acyl" refers to the groups H--C(O)--, alkyl-C(O)--,
substituted alkyl-C(O)--, alkenyl-C(O)--, substituted
alkenyl-C(O)--, cycloalkyl-C(O)--, substituted cycloalkyl-C(O)--,
cycloalkenyl-C(O)--, substituted cycloalkenyl-C(O)--, aryl-C(O)--,
substituted aryl-C(O)--, heteroaryl-C(O)--, substituted
heteroaryl-C(O)--, heterocyclic-C(O)--, and substituted
heterocyclic-C(O)--. Acyl includes the "acetyl" group
CH.sub.3C(O)--.
[0107] "Acylamino" refers to the groups --NR.sup.47C(O)alkyl,
--NR.sup.47C(O)substituted alkyl, --NR.sup.47C(O)cycloalkyl,
--NR.sup.47C(O)substituted cycloalkyl, --NR.sup.47C(O)cycloalkenyl,
--NR.sup.47C(O)substituted cycloalkenyl, --NR.sup.47C(O)alkenyl,
--NR.sup.47C(O)substituted alkenyl, --NR.sup.47C(O)aryl,
--NR.sup.47C(O)substituted aryl, --NR.sup.47C(O)heteroaryl,
--NR.sup.47C(O)substituted heteroaryl, --NR.sup.47C(O)heterocyclic,
and --NR.sup.47C(O)substituted.
[0108] "Acyloxy" refers to the groups alkyl-C(O)O--, substituted
alkyl-C(O)O--, alkenyl-C(O)O--, substituted alkenyl-C(O)O--,
aryl-C(O)O--, substituted aryl-C(O)O--, cycloalkyl-C(O)O--,
substituted cycloalkyl-C(O)O--, cycloalkenyl-C(O)O--, substituted
cycloalkenyl-C(O)O--, heteroaryl-C(O)O--, substituted
heteroaryl-C(O)O--, heterocyclic-C(O)O--, and substituted
heterocyclic-C(O)O--.
[0109] "Amino" refers to the group --NH.sub.2.
[0110] "Substituted amino" refers to the group --NR.sup.48R.sup.49
where R.sup.48 and R.sup.49 are independently selected from the
group consisting of hydrogen, alkyl, substituted alkyl, alkenyl,
substituted alkenyl, aryl, substituted aryl, cycloalkyl,
substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl,
heteroaryl, substituted heteroaryl, heterocyclic, and substituted
heterocyclic, provided that R.sup.48 and R.sup.49 are both not
hydrogen. When R.sup.48 is hydrogen and R.sup.49 is alkyl, the
substituted amino group is sometimes referred to herein as
alkylamino. When R.sup.48 and R.sup.49 are alkyl, the substituted
amino group is sometimes referred to herein as dialkylamino. When
referring to a monosubstituted amino, it is meant that either
R.sup.48 or R.sup.49 is hydrogen but not both. When referring to a
disubstituted amino, it is meant that neither R.sup.48 nor R.sup.49
are hydrogen.
[0111] "Aminocarbonyl" refers to the group --C(O)NR.sup.50R.sup.51
where R.sup.50 and R.sup.51 are independently selected from the
group consisting of hydrogen, alkyl, substituted alkyl, alkenyl,
substituted alkenyl, aryl, substituted aryl, cycloalkyl,
substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl,
heteroaryl, substituted heteroaryl, heterocyclic, and substituted
heterocyclic and where R.sup.50 and R.sup.51 are optionally joined
together with the nitrogen bound thereto to form a heterocyclic or
substituted heterocyclic group, and wherein alkyl, substituted
alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl,
cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted
cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted
heteroaryl, heterocyclic, and substituted heterocyclic are as
defined herein.
[0112] "Aminothiocarbonyl" refers to the group
--C(S)NR.sup.50R.sup.51 where R.sup.50 and R.sup.51 are
independently selected from the group consisting of hydrogen,
alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,
substituted alkynyl, aryl, substituted aryl, cycloalkyl,
substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl,
heteroaryl, substituted heteroaryl, heterocyclic, and substituted
heterocyclic and where R.sup.50 and R.sup.51 are optionally joined
together with the nitrogen bound thereto to form a heterocyclic or
substituted heterocyclic group.
[0113] "Aminocarbonylamino" refers to the group
--NR.sup.47C(O)NR.sup.50R.sup.51 where R.sup.47 is hydrogen or
alkyl and R.sup.50 and R.sup.51 are independently selected from the
group consisting of hydrogen, alkyl, substituted alkyl, alkenyl,
substituted alkenyl, aryl, substituted aryl, cycloalkyl,
substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl,
heteroaryl, substituted heteroaryl, heterocyclic, and substituted
heterocyclic, and where R.sup.50 and R.sup.51 are optionally joined
together with the nitrogen bound thereto to form a heterocyclic or
substituted heterocyclic group.
[0114] "Aminocarbonyloxy" refers to the group
--O--C(O)NR.sup.50R.sup.51 where R.sup.50 and R.sup.51 are
independently selected from the group consisting of hydrogen,
alkyl, substituted alkyl, alkenyl, substituted alkenyl, aryl,
substituted aryl, cycloalkyl, substituted cycloalkyl, cycloalkenyl,
substituted cycloalkenyl, heteroaryl, substituted heteroaryl,
heterocyclic, and substituted heterocyclic and where R.sup.50 and
R.sup.51 are optionally joined together with the nitrogen bound
thereto to form a heterocyclic or substituted heterocyclic
group.
[0115] "Aryl" or "Ar" refers to a monovalent aromatic carbocyclic
group of from 6 to 14 carbon atoms having a single ring (e.g.,
phenyl) or multiple condensed rings (e.g., naphthyl or anthryl)
which condensed rings may or may not be aromatic (e.g.,
2-benzoxazolinone, 2H-1,4-benzoxazin-3(4H)-one-7-yl, and the like)
provided that the point of attachment is at an aromatic carbon
atom. Preferred aryl groups include phenyl and naphthyl.
[0116] "Substituted aryl" refers to aryl groups which are
substituted with 1 to 5, preferably 1 to 3, or more preferably 1 to
2 substituents selected from the group consisting of alkyl,
substituted alkyl, alkenyl, substituted alkenyl, alkoxy,
substituted alkoxy, acyl, acylamino, acyloxy, amino, substituted
amino, aminocarbonyl, aminocarbonylamino, aminocarbonyloxy,
aminosulfonyl, aminosulfonyloxy, aminosulfonylamino, aryl,
substituted aryl, aryloxy, substituted aryloxy, carboxyl, carboxyl
ester, (carboxyl ester)amino, (carboxyl ester)oxy, cyano,
cycloalkyl, substituted cycloalkyl, cycloalkyloxy, substituted
cycloalkyloxy, cycloalkenyl, substituted cycloalkenyl,
cycloalkenyloxy, substituted cycloalkenyloxy, halo, hydroxy,
heteroaryl, substituted heteroaryl, heteroaryloxy, substituted
heteroaryloxy, heterocyclic, substituted heterocyclic,
heterocyclyloxy, substituted heterocyclyloxy, and nitro.
[0117] "Arylene" refers to a divalent aromatic carbocyclic group of
from 6 to 14 carbon atoms having a single ring or multiple
condensed rings. "Substituted arylene" refers to an arylene having
from 1 to 5, preferably 1 to 3, or more preferably 1 to 2
substituents as defined for aryl groups.
[0118] "Aryloxy" refers to the group --O-aryl, where aryl is as
defined herein, that includes, by way of example, phenoxy and
naphthoxy.
[0119] "Substituted aryloxy" refers to the group --O-(substituted
aryl) where substituted aryl is as defined herein.
[0120] "Carbonyl" refers to the divalent group --C(O)-- which is
equivalent to --C(.dbd.O)--.
[0121] "Carboxyl" or "carboxy" refers to --COOH or salts
thereof.
[0122] "Carboxyl ester" or "carboxy ester" refers to the group
--C(O)(O)-alkyl, --C(O)(O)-substituted alkyl, --C(O)O-alkenyl,
--C(O)(O)-substituted alkenyl, --C(O)(O)-aryl,
--C(O)(O)-substituted-aryl, --C(O)(O)-cycloalkyl,
--C(O)(O)-substituted cycloalkyl, --C(O)(O)-cycloalkenyl.
--C(O)(O)-substituted cycloalkenyl, --C(O)(O)-heteroaryl,
--C(O)(O)-substituted heteroaryl, --C(O)(O)-heterocyclic, and
--C(O)(O)-substituted heterocyclic.
[0123] "(Carboxyl ester)amino refers to the group
--NR.sup.47C(O)(O)-alkyl, --NR.sup.47C(O)(O)-substituted alkyl,
--NR.sup.47C(O)O-alkenyl, --NR.sup.47C(O)(O)-substituted alkenyl,
--NR.sup.47C(O)(O)-aryl, --NR.sup.47C(O)(O)-substituted-aryl,
--NR.sup.47C(O)(O)-cycloalkyl, --NR.sup.47C(O)(O)-substituted
cycloalkyl, --NR.sup.47C(O)(O)-cycloalkenyl,
--NR.sup.47C(O)(O)-substituted cycloalkenyl,
--NR.sup.47C(O)(O)-heteroaryl, --NR.sup.47C(O)(O)-substituted
heteroaryl, --NR.sup.47C(O)(O)-heterocyclic, and
--NR.sup.47C(O)(O)-substituted heterocyclic wherein R.sup.47 is
alkyl or hydrogen, and wherein alkyl, substituted alkyl, alkenyl,
substituted alkenyl, cycloalkyl, substituted cycloalkyl,
cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl,
heteroaryl, substituted heteroaryl, heterocyclic, and substituted
heterocyclic are as defined herein.
[0124] "(Carboxyl ester)oxy refers to the group --O--C(O)O-alkyl,
--O--C(O)O-substituted alkyl, --O--C(O)O-alkenyl,
--O--C(O)O-substituted alkenyl, --O--C(O)O-aryl,
--O--C(O)O-substituted-aryl, --O--C(O)O-cycloalkyl,
--O--C(O)O-substituted cycloalkyl, --O--C(O)O-cycloalkenyl,
--O--C(O)O-substituted cycloalkenyl, --O--C(O)O-heteroaryl,
--O--C(O)O-substituted heteroaryl, --O--C(O)O-heterocyclic, and
--O--C(O)O-substituted heterocyclic wherein alkyl, substituted
alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl,
cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted
cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted
heteroaryl, heterocyclic, and substituted heterocyclic are as
defined herein.
[0125] "Cyano" refers to the group --CN.
[0126] "Cycloalkyl" refers to cyclic alkyl groups of from 3 to 10
carbon atoms having single or multiple cyclic rings including
fused, bridged, and spiro ring systems. The fused ring can be an
aryl ring provided that the non aryl part is joined to the rest of
the molecule. Examples of suitable cycloalkyl groups include, for
instance, adamantyl, cyclopropyl, cyclobutyl, cyclopentyl, and
cyclooctyl.
[0127] "Cycloalkenyl" refers to non-aromatic cyclic alkyl groups of
from 3 to 10 carbon atoms having single or multiple cyclic rings
and having at least one >C.dbd.C<ring unsaturation and
preferably from 1 to 2 sites of >C.dbd.C<ring
unsaturation.
[0128] "Substituted cycloalkyl" and "substituted cycloalkenyl"
refers to a cycloalkyl or cycloalkenyl group having from 1 to 5 or
preferably 1 to 3 substituents selected from the group consisting
of oxo, thioxo, alkyl, substituted alkyl, alkenyl, substituted
alkenyl, alkoxy, substituted alkoxy, acyl, acylamino, acyloxy,
amino, substituted amino, aminocarbonyl, aminocarbonylamino,
aminocarbonyloxy, aryl, substituted aryl, aryloxy, substituted
aryloxy, carboxyl, carboxyl ester, (carboxyl ester)amino, (carboxyl
ester)oxy, cyano, cycloalkyl, substituted cycloalkyl,
cycloalkyloxy, substituted cycloalkyloxy, cycloalkenyl, substituted
cycloalkenyl, cycloalkenyloxy, substituted cycloalkenyloxy, halo,
hydroxy, heteroaryl, substituted heteroaryl, heteroaryloxy,
substituted heteroaryloxy, heterocyclic, substituted heterocyclic,
heterocyclyloxy, substituted heterocyclyloxy, and nitro.
[0129] "Cycloalkyloxy" refers to --O-cycloalkyl.
[0130] "Substituted cycloalkyloxy refers to --O-(substituted
cycloalkyl).
[0131] "Cycloalkenyloxy" refers to --O-cycloalkenyl.
[0132] "Substituted cycloalkenyloxy" refers to --O-(substituted
cycloalkenyl).
[0133] "Guanidino" refers to the group --NHC(.dbd.NH)NH.sub.2.
[0134] "Halo" or "halogen" refers to fluoro, chloro, bromo and
iodo.
[0135] "Hydroxy" or "hydroxyl" refers to the group --OH.
[0136] "Heteroaryl" refers to an aromatic group of from 1 to 10
carbon atoms and 1 to 4 heteroatoms selected from the group
consisting of oxygen, nitrogen and sulfur within the ring. Such
heteroaryl groups can have a single ring (e.g., pyridinyl or furyl)
or multiple condensed rings (e.g., indolizinyl or benzothienyl)
wherein the condensed rings may or may not be aromatic and/or
contain a heteroatom provided that the point of attachment is
through an atom of the aromatic heteroaryl group. In one
embodiment, the nitrogen and/or the sulfur ring atom(s) of the
heteroaryl group are optionally oxidized to provide for the N-oxide
(N.fwdarw.O), sulfinyl, or sulfonyl moieties. Certain non-limiting
examples include pyridinyl, pyrrolyl, indolyl, thiophenyl,
oxazolyl, thizolyl, and furanyl.
[0137] "Substituted heteroaryl" refers to heteroaryl groups that
are substituted with from 1 to 5, preferably 1 to 3, or more
preferably 1 to 2 substituents selected from the group consisting
of the same group of substituents defined for substituted aryl.
[0138] "Heteroaryloxy" refers to --O-heteroaryl.
[0139] "Substituted heteroaryloxy" refers to the group
--O-(substituted heteroaryl).
[0140] "Heterocycle" or "heterocyclic" or "heterocycloalkyl" or
"heterocyclyl" refers to a saturated or partially saturated, but
not aromatic, group having from 1 to 10 ring carbon atoms and from
1 to 4 ring heteroatoms selected from the group consisting of
nitrogen, sulfur, or oxygen. Heterocycle encompasses single ring or
multiple condensed rings, including fused bridged and spiro ring
systems. In fused ring systems, one or more the rings can be
cycloalkyl, aryl, or heteroaryl provided that the point of
attachment is through a non-aromatic ring. In one embodiment, the
nitrogen and/or sulfur atom(s) of the heterocyclic group are
optionally oxidized to provide for the N-oxide, sulfinyl, or
sulfonyl moieties.
[0141] "Substituted heterocyclic" or "substituted heterocycloalkyl"
or "substituted heterocyclyl" refers to heterocyclyl groups that
are substituted with from 1 to 5 or preferably 1 to 3 of the same
substituents as defined for substituted cycloalkyl.
[0142] "Heterocyclyloxy" refers to the group --O-heterocycyl.
[0143] "Substituted heterocyclyloxy" refers to the group
--O-(substituted heterocycyl).
[0144] Examples of heterocycle and heteroaryls include, but are not
limited to, azetidine, pyrrole, furan, thiophene, imidazole,
pyrazole, pyridine, pyrazine, pyrimidine, pyridazine, indolizine,
isoindole, indole, dihydroindole, indazole, purine, quinolizine,
isoquinoline, quinoline, phthalazine, naphthylpyridine,
quinoxaline, quinazoline, cinnoline, pteridine, carbazole,
carboline, phenanthridine, acridine, phenanthroline, isothiazole,
phenazine, isoxazole, phenoxazine, phenothiazine, imidazolidine,
imidazoline, piperidine, piperazine, indoline, phthalimide,
1,2,3,4-tetrahydroisoquinoline,
4,5,6,7-tetrahydrobenzo[b]thiophene, thiazole, thiazolidine,
thiophene, benzo[b]thiophene, morpholinyl, thiomorpholinyl (also
referred to as thiamorpholinyl), 1,1-dioxothiomorpholinyl,
piperidinyl, pyrrolidine, and tetrahydrofuranyl.
[0145] "Nitro" refers to the group --NO.sub.2.
[0146] "Oxo" refers to the atom (.dbd.O).
[0147] The term "oxyalkylene" refers to --O-alkylene- and the term
"polyoxyalkylene" refers to "--(O-alkylene).sub.n where n is an
integer from 1 to 15.
[0148] Unless indicated otherwise, the nomenclature of substituents
that are not defined herein are arrived at by naming the terminal
portion of the functionality followed by the adjacent functionality
toward the point of attachment. For example, the substituent
"alkoxycarbonylalkyl" refers to (alkoxy)-C(O)-(alkyl)-. Likewise,
"alkarylene" refers to -alkylene-arylene- and "arylalkylene" refers
to -arylene-alkylene-.
[0149] It is understood that in all substituted groups defined
above, polymers arrived at by defining substituents with further
substituents to themselves (e.g., substituted aryl having a
substituted aryl group as a substituent which is itself substituted
with a substituted aryl group, etc.) are not intended for inclusion
herein. In such cases, the maximum number of such substituents is
three. That is to say that each of the above definitions is
constrained by a limitation that, for example, substituted aryl
groups are limited to -substituted aryl-(substituted
aryl)-substituted aryl.
[0150] It is understood that the above definitions are not intended
to include impermissible substitution patterns (e.g., methyl
substituted with 5 fluoro groups). Such impermissible substitution
patterns are well known to the skilled artisan.
[0151] "Tautomer" refer to alternate forms of a compound that
differ in the position of a proton, such as enol-keto and
imine-enamine tautomers, or the tautomeric forms of heteroaryl
groups containing a ring atom attached to both a ring --NH-- moiety
and a ring .dbd.N-- moiety such as pyrazoles, imidazoles,
benzimidazoles, triazoles, and tetrazoles. Fluorescein is an
example of a compound with tautomeric structures as depicted
below:
##STR00066##
Likewise, folic acid (as well as pteroic acid) is another example
of a compound with tautomeric structures as depicted below:
##STR00067##
It is understood that depiction of any tautomer structure herein is
intended to include their corresponding tautomers.
[0152] As used herein, the term stereochemically pure denotes a
compound which has 80% or greater by weight of the indicated
stereoisomer and 20% or less by weight of other stereoisomers. In a
further embodiment, the compounds of this invention have 90% or
greater by weight of the stated stereoisomer and 10% or less by
weight of other stereoisomers. In a yet further embodiment, the
compounds of this invention have 95% or greater by weight of the
stated stereoisomer and 5% or less by weight of other
stereoisomers. In a still further embodiment, the compounds of this
invention have 97% or greater by weight of the stated stereoisomer
and 3% or less by weight of other stereoisomers.
[0153] The term "fluorescent masking agent" refers to a group
covalently bound to a fluorescent moiety through a cleavable bond
that significantly limits or eliminates the ability of that
fluorescent moiety to fluoresce. That is to say that the signal
generating capacity of the fluorescent moiety has been reduced by
at least 90% or more and preferably completely eliminated. When so
modified, the masked fluorescent compound is sometimes referred to
herein as a "pro-fluorescent compound". Examples of such masking
include acylation of both hydroxyl groups of fluorescein and
derivatives thereof. When so acylated, the fluorescein and its
derivatives are locked into a structure that cannot fluoresce. Such
masking agents do not include quenchers whether associated with or
bound to a fluorophore as the underlying fluorophore still
generates a fluorescent signal but that signal is captured by the
quencher as opposed to masking the fluorophore by reducing or
eliminating its fluorescence.
[0154] The term "unmasked" refers to removal of the masking
agent(s) from the masked fluorescent imaging agent so that the
imaging agent can once again generate fluorescence. Unmasking
generally requires cleavage of a cleavable bond that couples the
masking agent to the conjugate and, preferably, to the imaging
agent.
[0155] The term "cleavable bond" refers to bonds that stable in the
extracellular domain but are cleaved in the intracellular domain of
cancer cells. Such cleavable bonds include glycosides, esters,
orthoesters, carbonates, and the like. Esters are a particularly
preferred cleavable bond.
[0156] The term "stable" refers to a bond that has a sufficient
half-life in physiological fluids including serum such that there
is preferential targeting of the pro-fluorescent moiety to the
cancer cell. One preferred half-life is at least about 2 minutes,
more preferably at least 5 minutes and even more preferably at
least 15 minutes.
[0157] The term "targeting agent" refers to any component or moiety
that binds with specificity to a cancer cell through a receptor
that permits absorption of such targeting agents into the cell. For
the purposes of this application, such targeting agents do not
include antibodies such as monoclonal antibodies as absorption into
such cell does not typically occur with antibodies. Examples of
targeting agents include folic acid, pteroic acid, folic acid alpha
C.sup.1-C.sub.4 alkyl ester, luteinizing hormone releasing hormone
(LHRH), Dharap, et al., PNAS, 120(36):12962-12967 (2005)
(incorporated herein by reference in its entirety),
avidin/streptavidin (binds to the D-galactose receptors commonly
found on cancer cells, see e.g. Ogawa et al. Mol. Pharm. 2009;
6(2): 386-395), and the like.
[0158] The term "fluorescent imaging agent" refers to imaging
agents that produce a fluorescent signal upon exposure to
ultraviolet light and which agents are capable of being masked by
covalent linkage of a moiety or moieties thereto provided that such
moiety or moieties do not constitute quenchers. For the sake of
completion, a "quencher" is a material that is associated with or
bound to a fluorescent compound or moiety that, because of its
proximity to the fluorescent compound or moiety, absorbs the
fluorescence generated by that compound or moiety. Such is in
contrast to a masking agent that prevents or reduces the
fluorescence generated by the fluorescent compound or moiety.
[0159] The term "conjugate" or "compound" refers to a molecule
comprising a targeting agent and a fluorescent imaging agent as
components (or moieties) of that compound or conjugate. The
fluorescent imaging agent is bound to the targeting agent via a
covalent bond. In one embodiment, the fluorescent imaging agent is
masked by ester group(s) that can readily be cleaved under
intracellular conditions.
[0160] The term "cleaved" refers to breakage of the cleavable bond
thereby regenerating the signal generating capacity of the
previously pro-fluorescent moiety.
[0161] The term "linker" refers to a linking group of from 1 to 30
carbon atoms in length optionally having from 1 to 15 heteroatoms
such as oxygen, oxo (.dbd.O), thioxo (.dbd.S), phosphorus
(including oxides thereof such as phosphite, phosphate, etc.),
sulfur (including oxides thereof such as sulfite and sulfate),
nitrogen (e.g., NR.sup.1 where R.sup.1 is as defined above, and the
like.) One preferred linker is a polyoxyalkylene of from 1 to 10
oxyalkylene units.
[0162] The term "detectible signal" refers to a signal that can be
generated by the fluorescent imaging agent that is detectible
either visually, spectroscopically, or by suitable software. It is
understood that fluorescent signaling requires exposure of cells to
an ultraviolet light source comprising the excitation wavelength of
the fluorescent imaging agent so as to produce fluorescence.
[0163] The compounds of this invention may exist as solvates,
especially hydrates. Hydrates may form during manufacture of the
compounds or compositions comprising the compounds, or hydrates may
form over time due to the hygroscopic nature of the compounds.
Compounds of this invention may exist as organic solvates as well,
including dimethylformamide, ether, and alcohol solvates among
others. The identification and preparation of any particular
solvate is within the skill of the ordinary artisan of synthetic
organic or medicinal chemistry.
[0164] "Subject" refers to a mammal. The mammal can be a human or
non-human animal mammalian organism.
[0165] "Treating" or "treatment" refers to the application of one
or more pro-fluorescent compounds of this invention onto mammalian
cells that are suspected of containing cancerous cells that express
or overexpress the folic acid receptor so as to allow the clinician
to confirm as to whether such cells are, in fact, such cancerous
cells.
[0166] "Effective amount" refers to the amount of a compound of
this invention that is sufficient to diagnostically evaluate
whether a cell is cancerous or not.
[0167] The term "specificity" refers to the specific accumulation
of a conjugate of this invention onto/into cancer cells at a rate
sufficiently higher than non-cancerous or normal cells such that
the fluorescence generated by such cancer cells is distinguishable
from normal cells. In one embodiment, the conjugates of this
invention accumulate at a rate of greater than 2:1 in cancer cells
compared to non-cancerous cells. Preferably, the rate of
accumulation is greater than 3:1, more preferably greater than 5:1,
and even more preferably greater than 6:1 and most preferably at
least 8:1. Accordingly the term "absorbed" refers to the surface
accumulation or intracellular accumulation of the conjugates of
this invention, although intracellular accumulation is
desirable.
[0168] As used herein, the term "salts" of compounds disclosed
herein are within the scope of the present invention include acid
or base addition salts which retain the desired activity. When the
compound of the present invention has a basic group, such as, for
example, an amino group, salts can be formed with inorganic acids
(such as hydrochloric acid, hydroboric acid, nitric acid, sulfuric
acid, and phosphoric acid), organic acids (e.g., alginate, formic
acid, acetic acid, benzoic acid, gluconic acid, glucuronic acid,
fumaric acid, oxalic acid, tartaric acid, lactic acid, maleic acid,
citric acid, succinic acid, malic acid, methanesulfonic acid,
benzenesulfonic acid, naphthalene sulfonic acid, and
p-toluenesolfonic acid) or acidic amino acids (such as aspartic
acid and glutamic acid). When the compound of the present invention
has an acidic group, such as for example, a carboxylic acid group,
it can form salts with metals, such as alkali and earth alkali
metals (e.g., Na.sup.+, Li.sup.+, K.sup.+, Ca.sup.2+, Mg.sup.2+,
Zn.sup.2+), ammonia or organic amines (e.g., dicyclohexylamine,
trimethylamine, trimethylamine, pyridine, picoline, ethanolamine,
diethanolamine, triethanolamine) or basic amino acids (e.g.,
arginine, lysine, and ornithine). Such salts can be prepared in
situ during isolation and purification of the compounds or by
separately reacting the purified compound in its free base or free
acid form with a suitable acid or base, respectively, and isolating
the salt thus formed.
[0169] Other fluorescent agents suitable for use in this invention
are well known in the art some of which are depicted below. These
agents can be modified into masked fluorescent agents that can be
conjugated to folic or pteroic acid.
##STR00068##
6. COMPOUNDS OF THIS INTENTION
[0170] This invention includes conjugates of the formula IA and IB:
a conjugate of the formula IA and IB:
##STR00069##
[0171] where L.sup.1 is a bond or a linker,
[0172] X is a masked imaging agent, and
[0173] Y is --O-- or >NR.sup.1 where R.sup.1 is hydrogen,
C.sub.1-C.sub.4 alkyl, substituted C.sub.1-C.sub.4 alkyl;
C.sub.2-C.sub.5 alkenyl, substituted C.sub.2-C.sub.5 alkenyl, aryl,
substituted aryl, cycloalkyl, substituted cycloalkyl, heteroaryl,
substituted heteroaryl, heterocyclic, substituted heterocyclic;
[0174] R.sup.2 is hydrogen or C.sub.1-C.sub.4 alkyl;
[0175] or salts, tautomers and/or solvates thereof,
[0176] provided that the conjugate is not
##STR00070##
[0177] wherein X'' is selected from the group consisting of --O--
and >NR.sup.20, L'' is a bond or linking group having 1 to 30
atoms, R'' is an alkyl group or a substituted alkyl group having
from 4 to 30 carbon atoms and optionally 1 to 6 heteroatoms
selected from oxygen, hydroxyl, sulfur, carbonyl, thiocarbonyl,
chloro, bromo, iodo, and cyano; and R.sup.20 is hydrogen or alkyl;
or
##STR00071##
where R.sup.11 and R.sup.10 are C.sub.1-C.sub.30 ester groups;
or
##STR00072##
where R is an alkyl group or a substituted alkyl group having from
4 to 30 carbon atoms and optionally 1 to 6 heteroatoms selected
from oxygen, hydroxyl, sulfur, thiol, carbonyl, thiocarbonyl,
chloro, bromo, iodo, and cyano; or
##STR00073##
[0178] wherein R.sup.40 is C.sub.1-C.sub.6 alkyl or heteroalkyl
optionally substituted with a water solubility imparting functional
group selected from the group consisting of hydroxy, methoxy,
amino, carboxylic acid, and carboxylate;
[0179] R.sup.45 is hydrogen or an optionally substituted alkyl
group;
[0180] L'' is a bond or a linking group having 1 to 30 atoms
including --NHC(O)-- alkylene-, --NHC(O)NH-alkylene-,
--NHC(S)NH-alkylene-, and --OC(O)NH--;
[0181] folate is a folic acid moiety joined with the rest of the
conjugate via an ester of the folic acid carboxyl group; and
[0182] folamide is a folic acid moiety joined with the rest of the
conjugate via an amide of the folic acid carboxyl group.
7. SYNTHESIS
General Synthetic Methods
[0183] The compounds of this invention can be prepared from readily
available starting materials using the following general methods
and procedures. It will be appreciated that where typical or
preferred process conditions (i.e., reaction temperatures, times,
mole ratios of reactants, solvents, pressures, etc.) are given,
other process conditions can also be used unless otherwise stated.
Optimum reaction conditions may vary with the particular reactants
or solvent used, but such conditions can be determined by one
skilled in the art by routine optimization procedures.
[0184] Additionally, as will be apparent to those skilled in the
art, conventional protecting groups may be necessary to prevent
certain functional groups from undergoing undesired reactions.
Suitable protecting groups for various functional groups as well as
suitable conditions for protecting and deprotecting particular
functional groups are well known in the art. For example, numerous
protecting groups are described in T. W. Greene and P. G. M. Wuts,
Protecting Groups in Organic Synthesis, Third Edition, Wiley, New
York, 1999, and references cited therein.
[0185] If the compounds of this invention contain one or more
chiral centers, such compounds can be prepared or isolated as pure
stereoisomers, i.e., as individual enantiomers or d(l) stereomers,
or as stereoisomer-enriched mixtures. All such stereoisomers (and
enriched mixtures) are included within the scope of this invention,
unless otherwise indicated. Pure stereoisomers (or enriched
mixtures) may be prepared using, for example, optically active
starting materials or stereoselective reagents well-known in the
art. Alternatively, racemic mixtures of such compounds can be
separated using, for example, chiral column chromatography, chiral
resolving agents and the like.
[0186] The starting materials for the following reactions are
generally known compounds or can be prepared by known procedures or
obvious modifications thereof. For example, many of the starting
materials are available from commercial suppliers such as Aldrich
Chemical Co. (Milwaukee, Wis., USA), Bachem (Torrance, Calif.,
USA), Emka-Chemce or Sigma (St. Louis, Mo., USA). Others may be
prepared by procedures, or obvious modifications thereof, described
in standard reference texts such as Fieser and Fieser's Reagents
for Organic Synthesis, Volumes 1-15 (John Wiley, and Sons, 1991),
Rodd's Chemistry of Carbon Compounds, Volumes 1-5, and
Supplementals (Elsevier Science Publishers, 1989). Organic
Reactions, Volumes 1-40 (John Wiley, and Sons, 1991), March's
Advanced Organic Chemistry, (John Wiley, and Sons, 5.sup.th
Edition, 2001), and Larock's Comprehensive Organic Transformations
(VCH Publishers Inc., 1989).
8. SYNTHESIS OF REPRESENTATIVE COMPOUNDS OF THE INVENTION
[0187] In one embodiment, the method involves reacting a folic acid
or pteroic acid with a fluorescent or pro-fluorescent (masked)
compound. If a fluorescent compound is used, it must be
post-treated after coupling to folic or pteroic acid to convert
that compound to a pro-fluorescent or masked compound. Generally,
however, it is preferred to combine a pro-fluorescent or masked
compound having a reactive functionality that will form a covalent
bond with folic or pteroic acid either directly or through a
suitable linker.
[0188] In particular, the pro-fluorescent compounds used in the
synthesis of conjugates of this invention are readily converted
from fluorescent compounds using conventional methods. In one
illustrative embodiment, fluorescein 5-isothiocyanate is converted
to the corresponding diester via conventional esterification
conditions as depicted below:
##STR00074##
where each R.sup.8 is independently a C.sub.2-C.sub.20 group
optionally containing 1 to 10 heteroatoms selected from --O--,
--S--, carbonyl, >NR.sup.1, --NR.sup.1R.sup.1, --OH, --SH,
phosphate and sulfate. In particular, commercially available
fluorescein 5-isothiocyanate, compound 1, is combined with at least
two equivalents and preferably an excess of a suitable carboxylic
acid or acid anhydride in the presence of an excess of
N,N-dicyclohexylcarbodiimide (DCC) and 4-dimethylaminopyridine
(DMAP) in a suitable solvent or solvent mixture such as
N,N-dimethylformamide/pyridine. The hydroxyl groups of fluorescein
are readily esterified under such conditions. The reaction is
typically conducted at a temperature of from about 0.degree. to
about 60.degree. C. for a period of time sufficient to allow for
substantial completion of the reaction. Typically, such reactions
are conducted for about 1 to 24 hours. Afterwards, the resulting
product is recovered by conventional methods such as
crystallization, column chromatography, HPLC, or precipitation.
Reaction completion can be readily analyzed by loss of fluorescence
in the reaction mixture.
[0189] It is understood that while compound 1 is illustrated as
fluorescein 5-isothiocyanate, this compound is often a mixture with
fluorescein 6-isothiocyanate and that the two compounds are deemed
equivalents. As such, any reference to fluorescein 5-isothiocyanate
is meant to include fluorescein 6-isothiocyanate. Likewise, the
term "fluorescein" is intended to cover all known substitutions on
the ring system for fluorescein such at dichloro substituted
fluorescein compounds.
[0190] Separately, folic acid is converted to folic anhydride using
methods well known in the art as described by Guaragna, et al,
Bioconjugate Chemistry, 2012, 23:84-96 and especially at page 88
and as depicted below in Scheme 2. Specifically, folic acid,
compound 3 is combined DCC in a solvent mixture of DMF
(dimethylformamide) and pyridine (5:1). The reaction is conducted
at a slightly elevated temperature of about 30.degree. C. although
the reaction can be run at from about 0.degree. to about 60.degree.
C. The reaction is continued until substantially complete and the
product, folic anhydride--compound 4, can be recovered by
conventional means such as chromatography, distillation,
precipitation, high performance liquid chromatography (HPLC) and
the like. Alternatively, the product can be used in the next step
without purification and/or isolation.
##STR00075##
[0191] Folic anhydride, compound 4, is next ring opened to form an
amide linker moiety, compound 5, as shown in Scheme 3 below:
##STR00076##
[0192] Specifically, commercially available
4-aminomethyl-N-Boc-aniline is combined with folic anhydride,
compound 4, together with dicyclohexylcarbodiimide (DCC) under
conditions also set forth by by Guaragna, et al., Bioconjugate
Chemistry, 2012, 23:84-96 to provide for compound 5. Removal of the
Boc (t-butoxycarbonyl) protecting group proceeds via conventional
methods using trifluoroacetic acid to provide for the free amino
group (compound 6-Scheme 4). The reaction is continued until
substantially complete and the product, compound 4, can be
recovered by conventional means such as chromatography,
distillation, precipitation, HPLC, and the like. Alternatively, the
product is used in the next step without purification and/or
isolation.
[0193] In Scheme 3, the optional inclusion of a C1-C4 alcohol such
as methanol leads to the corresponding ester.
[0194] Compound 6 is next linked to compound 2 to form compound
(conjugate) 7 as shown in Scheme 4 below:
##STR00077##
[0195] Specifically, compound 2 and compound 6 are combined in a
suitable inert aprotic solvent such as dimethylformamide (DMF),
acetonitrile, methylene chloride, chloroform, ethyl acetate,
tetrahydrofuran and the like. The reaction is typically conducted
at from about 0.degree. to about 50.degree. C. for period of time
sufficient to substantially complete the reaction and preferably 1
to 24 hours. The reaction completion can be monitored by thin layer
chromatography (TLC), high performance liquid chromatography
(HPLC), and the product can be recovered by conventional methods
such as chromatography, precipitation, crystallization, HPLC, and
the like.
[0196] Alternatively, pteroic acid can be used in place of folic
acid in the above reaction. The reaction conditions are
substantially the same and the product is recovered in
substantially the same manner. Note that the carboxyl group of
pteroic acid will react similarly to the gamma carboxyl group of
folic acid.
[0197] In the reaction schemes above, fluorescein based compounds
can be replaced by non-fluorescein based compounds provided that
such compounds are capable of having their fluorescence
significantly reduced or eliminated by masking groups.
[0198] Still further, linkage of the folic acid to fluorescein can
be accomplished through ester bond formation off of one of the
phenolic alcohols as shown below:
##STR00078##
where R.sup.2, R.sup.50, and L.sup.1 are as defined above.
[0199] Specifically, in the reaction above, folic anhydride
(described above) is combined with fluorescein monoester wherein
the remaining hydroxyl group is retained or extended via a linker
using conventional techniques. In one embodiment, the linker is a
polyoxyalkylene chain of from 1 to 20 units and, in one embodiment,
that chain can be represented from left to right as
(CH.sub.2CH.sub.2O).sub.l where l is an integer of from 1 to 20.
Introduction of such polyoxyalkylene chains or other linkers is
well known in the art. Formation of the ester is also well known in
the art and is described above albeit the formation of a sodium
phenoxide derivative prior to ester formation will facilitate the
reaction. Conversion of the alpha carboxyl group to an ester again
proceeds via well known chemistry. In addition, the use of pteroic
acid in this reaction in place of folic acid proceeds as above.
9. METHODS AND COMPOSITIONS
[0200] The compounds of this invention are useful for detecting
cancer cells in a cellular sample suspected of containing cancer
cells and, preferably, cancer cells that overexpress folic acid
receptors. Many cancers overexpress these receptors including but
not limited to ovarian, cervical, epithelial, breast, colorectal,
brain, lung, and kidney (renal) cancers. These compounds are
applied to a cellular sample suspected of containing cancer cells
such as those recited above. Application can be made via any
conventional route such as intermixing the cellular sample with a
liquid formulation of one or more compounds of this invention. For
example, a sterile aqueous solution of a conjugate of this
invention can be sprayed onto a cellular mass such as a surgical
site or "painted" onto such a site. Alternatively, the compositions
can be targeted to specific sites of the surgical field that the
clinician wishes to evaluate for cancer cells.
[0201] In particular, the compounds/conjugates of this invention
uniquely involve a masking group that blocks the label portion of
the conjugate from signaling. The masking group is rapidly removed
when introduced into cancer cells but remains intact in the
extracellular space. In one embodiment, the label portion is a
fluorescein compound or derivative thereof and the masking groups
are esters on each of the alcohol groups on the fluorescein.
Without being limited to any theory, blocking of the signalizing of
these fluorescein compounds arises because esterification locks
these compounds into a structure that is incapable of generating a
fluorescent signal. Still further, once absorbed, intracellular
esterases and other enzymes remove these ester functionalities on
the label thereby restoring its labeling properties. Moreover,
these conjugates exhibit directional capacity to selectively bind
to and be absorbed by folate dependent cancers. That is to say that
the folic acid or pteroic acid moiety on the conjugates allow for
selective binding to cancer cells that have folic acid receptors
and in particular excess folic acid receptors as compared to normal
cells.
[0202] When so used, the compounds of this invention will be
administered in a diagnostically effective amount by any of the
accepted modes of administration for agents that serve similar
utilities. The actual amount of the compound of this invention,
i.e., the active ingredient, will depend upon numerous factors such
as the size of the surgical site of the subject, the signal
strength of the unmasked compound used, the route and form of
application, and other factors well-known to the skilled artisan.
In one particular embodiment, the surgical oncologist can reapply
the compound/compositions described herein multiple times in order
to identify remnant cancer cells.
[0203] This invention is not limited to any particular composition
or carrier, as such may vary. In general, compounds of this
invention will be administered as aqueous compositions. Such
compositions comprise water and optionally a biologically
compatible amount of a cosolvent such as ethanol, DMSO, ethyl
lactate, and the like. For example, a solvent mixture of 9:1 water:
DMSO provides a suitable solution of a compound of this invention
to be applied to cells or tissues. In one embodiment, such
compositions comprise from about 0.00001 to about 90 weight percent
of a compound of this invention and preferably about 0.001 to about
10 weight percent and even more preferably from about 0.001 to 1
weight percent.
[0204] Suitable compositions comprising compounds of this invention
may be manufactured by any of the methods well-known in the art,
such as, for example, by conventional mixing, dissolving, melting,
emulsifying, and the like.
10. FORMULATION EXAMPLES
[0205] The following are representative pharmaceutical formulations
containing a compound of this invention.
Formulation Example 1--Suspension Formulation
[0206] The following ingredients are mixed to form a suspension for
oral administration.
TABLE-US-00005 Ingredient Amount compound of this invention 10 mg
colorings 0.5 mg distilled water q.s. to 100 mL
Formulation Example 2--Injectable Formulation
[0207] The following ingredients are mixed to form an injectable
formulation.
TABLE-US-00006 Ingredient Amount compound of this invention 0.2
mg-20 mg sodium acetate buffer solution, 0.4M 2.0 mL HCl (1N) or
NaOH (1N) q.s. to suitable pH water (distilled, sterile) q.s. to 20
mL
11. EXAMPLES
[0208] This invention is further understood by reference to the
following examples, which are intended to be purely exemplary of
this invention. Any methods that are functionally equivalent are
within the scope of this invention. Various modifications of this
invention in addition to those described herein will become
apparent to those skilled in the art from the foregoing description
and accompanying figures. Such modifications fall within the scope
of the appended claims.
[0209] The following abbreviations are used in the examples below
and have the following meanings. If an abbreviation is not defined,
it has its art recognized meaning. In addition, all temperatures
are in degrees Celcius unless otherwise noted.
[0210] DCC=dicyclohexycarbodiimide
[0211] DMF=N,N-dimethylformamide
[0212] DMAP=N,N-dimethylaminopyridine
[0213] DMSO=dimethylsulfoxide
[0214] eq.=equivalents
[0215] ether=diethyl ether
[0216] FBS=fetal bovine serum
[0217] mg=milligram
[0218] mL=milliliter
[0219] mm=millimeter
[0220] mM=millimolar
[0221] mmoles=millimoles
[0222] RPMI=Roswell Park Memorial Institute medium
[0223] MP=melting point
[0224] RT=room temperature
[0225] TFA=trifluoroacetic acid
[0226] TLC=thin layer chromatography
[0227] .mu.M=micromolar
[0228] .mu.m=micromoles
[0229] V/V=volume/volume
Example 1--Synthesis of
2-(4-(((2-amino-4-oxo-3,4dihydropteridin-6-yl)methyl)amino)benzamido)-5-(-
(3'6'-dihydroxy-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthen]-5-yl)amino-5-ox-
opentanoic acid (compound 13)
##STR00079##
[0231] A mixture of 100 mg (0.22 mmoles) folic acid (compound 10)
in an anhydrous 20 mL DMF solution plus 4 mL pyridine was heated
and vigorously shaken to get a clear golden solution. To this
solution was added 6 equivalents of DCC (280 mg, 1.36 mmoles). The
reaction mixture was mixed in an ultrasound bath in the dark for 15
minutes twice, while the bath warmed to about 29.degree. C. The
resulting cloudy solution was added to a flask containing 1.1
equivalents 5-aminofluorescein (86.5 mg, 0.249 moles) (compound
11). The resulting reaction mixture was wrapped with aluminum foil
and stirred at room temperature overnight. After about 18 hours the
mixture was filtered through celite and added drop-wise to a
mixture of 70 mL ether and 30 mL acetone. The cloudy mixture was
stored in the dark in a freezer for several days. The solid
(compound 12) was filtered off, washed with ether and air-dried to
a constant mass of 115.7 mg (66.3%).
[0232] 29.4 mg of the folic acid-fluorescein conjugate (compound
12) was dissolved in 2 mL dry DMF, and 6 equivalents (23.6 mg)
triethylamine was added, followed by 5 equivalents of propionyl
chloride (17.6 mg). The reaction was stirred at room temperature
for several days, and poured into a mixture of water and ethyl
acetate. The organic layer was washed with water and brine and
dried over sodium sulfate. After evaporation of the solvent the
solid was further dried to yield 20.6 mg (54%). MP:
>290.degree.. Confirmation of the product was further provided
by loss of fluorescence due to the diesterification of the
fluorescein phenolic hydroxyl groups of compound 13. Specifically,
when a sample of compound 13 was dissolved in methanol a
non-fluoresecent solution resulted. On treatment with a few drops
of ammonia water intense fluorescence was noted. The ammonia is a
strong deacylating agent that unmasks the masked fluorescent
fluorescein diester.
Example 2--Synthesis of
O,O'-(5-(3-(4-((4-(4-(((2-amino-4-oxo-3,4-dihydropteridin-6-yl)methyl)ami-
no)benzamido)-5-methoxy-5-oxopentanamido)methyl)phenyl)thioureido)-3-oxo-3-
H-spiro[isobenzofuran-1,9'-xanthene]-3',6'-diyl)
bis)(2-methoxyethyl) disuccinate (compound 18)
##STR00080## ##STR00081##
[0233] A. Synthesis of
O,O'-5-isothiocyanato-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-3',6'-d-
iyl) bis(2-methoxyethyl)disuccinate (compound 17)
##STR00082##
[0235] The above reaction follows the literature preparation
described by J. Materials Chemistry, 2014, 2(26):4142-4145.
Specifically, a slight excess of succinic anhydride was combined
with 2-methoxyethanol in methylene chloride in a flask at about
20.degree. C. A solution of triethylamine in methylene chloride was
added dropwise over about a 15 minute period during which the
reaction produced sufficient heat so that the solvent began to
boil. Afterwards, the addition of triethylamine was stopped and the
reaction stirred overnight after returning to room temperature.
[0236] The reaction was stopped and the reaction solution washed
with brine and the organic layer was recovered. The solvent was
stripped and the resulting product was purified by column
chromatography (silica gel using a gradient of from 0 to 10%
methanol in methylene chloride v/v). The resulting product
(compound 12) was used as is without further purification or
isolation.
##STR00083##
[0237] Approximately 1 eq. of compound 22 was dissolved in
methylene chloride and then combined with approximately 1 eq. of
DCC at room temperature. The mixture was stirred for approximately
5 minutes and then 0.25 equivalents of DMAP and approximately 0.25
eq. of fluorescein were added thereto. The reaction mixture was
then sonicated at 26.degree. C. until the suspension was
substantially dissipated which occurred over approximately 15
minutes. The resulting reaction mixture was stirred overnight at
room temperature and monitored for reaction completion by TLC. Upon
substantial reaction completion, the non-soluble components were
filtered and the resulting solution was placed on a silica column
for purification purposes. The column was eluted with a solvent
gradient starting at 0% methanol and 100% methylene chloride and
finishing with 10% methanol and 90% methylene chloride (v/v). The
elutant containing the desired compound was stripped of solvent and
the resulting compound 14 was substantially free of fluorescence
indicative of formation of diester. A small aliquot of the compound
was contacted with a sodium hydroxide solution that immediately
provided for fluorescence indicative of deacylation. The sodium
hydroxide is a strong deacylating agent that unmasks the masked
fluorescent fluorescein diester.
[0238] A mixture of 100 mg (0.22 mmoles) folic acid (compound 10)
in an anhydrous 20 mL DMF solution plus 4 mL pyridine was heated
and vigorously shaken to get a clear golden solution. To this
solution was added 6 equivalents of DCC (280 mg, 1.36 mmoles). The
reaction mixture was mixed in an ultrasound bath in the dark for 15
minutes twice, while the bath warmed to 29.degree. C. The resulting
cloudy solution was added to a flask containing 1.1 eq. tert-butyl
(4-aminomethyl)phenyl)-carbamate (58.3 mg) (compound 15). The
resulting reaction mixture was wrapped with aluminum foil and
stirred at room temperature overnight. After about 18 hours 1 mL of
methanol was added to esterify the alpha-carboxylic acid (this is
an optional step). After stirring for an additional 24 hours at RT,
the mixture was filtered through celite and added drop-wise to a
mixture of 70 mL ether and 30 mL acetone. The cloudy mixture was
stored in the dark in a freezer for several days. The solid was
filtered off, washed with ether and air-dried to a constant mass of
72 mg (48.2%). Without further purification, 24 mg of this compound
was added to 0.5 mL TFA (large excess) stirred at room temperature
and then stored in the refrigerator. The TFA was evaporated to
yield 20 mg (35.7 .mu.m) of compound 16. This material was then
dissolved in a small amount of DMF and treated with 1 equivalent
(25 mg) of compound 17. The reaction was stirred at room
temperature overnight. The mixture was added to a large amount of
ether (75 mL) to yield a precipitate, which was filtered off,
washed with ether and dried to 29 mg (64%) of a tan solid, MP:
softens at 200.degree. C. and melts at 216-226.degree. C. (compound
18) A small sample of this material was dissolved in methanol
yielding a clear colorless solution. When a few drops of ammonia
water were added, the solution became intensely fluorescent. The
ammonia is a strong deacylating agent that unmasks the masked
fluorescent fluorescein diester.
Comparative Example A
##STR00084##
[0240] Compound 25 was prepared following the procedures set forth
above ad provided for the title compound as a comparative example
(no ester groups on the fluorescein moiety).
Example 3--Synthesis of the Fluorescein Diester, Compound 26
##STR00085##
[0242] Following the procedure of Example 2 and omitting the
addition of methanol, compound 20 is prepared. When a sample of
compound 13 was dissolved in methanol, a non-fluoresecent solution
resulted. On treatment with a few drops of ammonia water intense
fluorescence was noted. The ammonia is a strong deacylating agent
that unmasks the masked fluorescent fluorescein diester.
Example 4--Synthesis of the Fluorescein Diester, Compound 27
##STR00086##
[0244] Compound 27 was prepared following the procedures set forth
above. Specifically, when a sample of compound 13 was dissolved in
methanol a non-fluoresecent solution resulted. On treatment with a
few drops of ammonia water intense fluorescence was noted. The
ammonia is a strong deacylating agent that unmasks the masked
fluorescent fluorescein diester.
Example 5--Synthesis of Pteroic Acid Derivative
##STR00087##
[0246] 12 mg of pteroic acid (Sigma Aldrich, St. Louis, Mo., USA)
was mixed with about 1.5 mL of DMF, and heated to about 80.degree.
C. with stirring for a few minutes. The pteroic acid did not go
into solution. The mixture was then cooled to RT and excess thionyl
chloride was added and a clear solution was generated almost
immediately indicating the formation of the acid chloride. 8.2 mg
4-(aminomethyl) t-Boc-aniline was added and the clear solution
stirred overnight. The clear golden-colored solution was treated
with a few drops of triethylamine and the solution became very dark
and viscous. After stirring for 3 hours, the solution was added to
50 mL of ether using several milliliters of acetone in the
transfer. The precipitate was filtered and was dark and wet. It was
treated with charcoal and washed through the funnel with DMF and
then acetone. The filtrate was again added to 50 mL of ether and a
lighter colored precipitate formed. The mixture was stored in a
refrigerator overnight. The solid was filtered off and treated with
excess trifluoroacetic acid (TFA) to remove the Boc group.
Dichloromethane was used as the solvent. The excess TFA was removed
under vacuum and the residue was dissolved in dichloromethane and
excess triethylamine added until the solution was basic. To this
solution was added excess fluorescein 5-isothiocyanate diester (as
depicted in the scheme above) in dichloromethane. The reaction was
then stirred at room temperature. The solution was then added to 10
mL of ether that was then cooled in ice. The solid was collected
after centrifuging and transferring to a glass fritted filter
funnel, washed with excess ether, and air-dried. About 8 mg of the
product was obtained as a dark solid.
MP=155-158.degree. C.
[0247] To confirm that the product contained the fluorescein
diester moiety, a sample of the product was dissolved in methanol
to provide a clear amber solution with no evidence of fluorescein
fluorescence. Upon addition of aqueous ammonia, an intense
characteristic yellow-green fluorescence was obtained.
Biological Examples
A. Detection of Ovarian Cancer Cells
[0248] Compound 24 and comparative compound A were evaluated for
their ability to be absorbed by cancer cells and then, in the case
of compound 24, deacylated by intracellular esterases so as to
regenerate a fluorescent structure. Specifically, approximately
500,000 SDOV3 cells (an ovarian cancer cell line) were seeded into
separate 35 mm culture dishes containing a folate-free growth
medium (RPMI+10% FBS). The next day, the medium was replaced with a
folate-free medium (no FBS). In one culture dish, the medium was
supplemented with 25 micromolar of compound 24; and, in another
culture dish, the medium was supplemented with 50 micromolar of
comparative compound A. After incubation, the cells were washed
with HBSS (Hank's balanced salt solution) to remove unbound
compound. The cells were then imaged with a 20.times. immersion
objective on a standard upright fluorescent microscope. In the case
of compound 24, the fluorescent signal was clear, consistent and
unambiguous evidencing that cancer cells were fluorescent and that
the fluorescent signal was not evident in the solution. FIG. 1
illustrates a picture showing the fluorescence generated. Note that
only the cancer cells evidenced fluorescence and that the solution
remained non-fluorescent. As to comparative compound A, that
solution showed a burst of fluorescence but immediately the
fluorescence was bleached and the composition no longer was capable
of fluorescing. This evidences that that composition was not
suitable for use in the methods described herein.
[0249] These results establish that compound 24 targeted cancer
cells, were absorbed by cancer cells, and were deacylated by
intracellular enzymes. The persistent signaling solely in the
cancer cells evidenced that deacylated compound 24 did not efflux
from the cancer cells. On the other hand, comparative compound A
also was absorbed by the cancer cells and immediately fluoresced
but that was followed by loss of fluorescence likely due to
bleaching under the intense light used.
[0250] Taken together, the compounds of this invention are suitable
for use in detecting remnant cancer cells. Because certain cancer
cells preferentially uptake the conjugates of this invention, after
incubation for a period of time, removal of the applied solution
from the surgical field will limit absorption into normal cells.
Such can be accomplished under conventional lavage/washing
conditions.
B. Detection of Ovarian Cancer Cells is Via the Folic Acic
Receptor
[0251] This example establishes that compound 24 is specific for
the folate receptor. Specifically, compound 24 was used in a folate
free medium and in a medium using excess folate that competed with
compound 24. The rationale is that in the presence of excess
folate, compound 24 would have to compete for binding to the folate
binding protein and therefore there would be less signal than when
compound 24 was the sole source of folate.
[0252] To test this hypothesis, SKOV3 cells were incubated with 10
PIM of compound 24 or with 10 .mu.M of compound 24+1 mM folate (100
xs). Cells were then washed and imaged as before.
[0253] All images were analyzed using exactly the same parameters.
Mean intensity was measured from identical regions with in each
image. Images, shown in FIGS. 2A and 2B, clearly indicate that
folate competes with compound 24 for labeling ovarian cancer cells.
These images establish that compound 24 binds to cells through the
folate binding protein and not by some other route.
C. Dose Response
[0254] This experiment establishes that compound 24 provides a dose
response relative to the fluorescence generated. Specifically,
SKOV3 cells were incubated with 10 .mu.M, 25 .mu.M, or 50 .mu.M of
compound 24 in RPMI supplemented with 0.25% BSA. Cells were
incubated for 1 hour then washed with HBSS and imaged as
before.
[0255] All images were analyzed using exactly the same parameters
using the image J software suite. Mean intensity was measured from
identical regions with in each image. Images, shown below, show a
clear dose response for compound 124 in labeling cells. The bar
graph shows the mean intensity of each image and supports the
conclusions from visual inspection of the images. While BSA was
used as a supplement, acylated BSA may be more practical.
[0256] The results of this experiment are provided below:
TABLE-US-00007 10 .mu.M 25 .mu.M 50 .mu.M Mean 312.8 360.4 545.5
Standard error 2.3 9.3 1.6
These results demonstrate a dose dependent response.
* * * * *